Báo cáo Y học: Expression of recombinant murine pregnancy-associated plasma protein-A (PAPP-A) and a novel variant (PAPP-Ai) with differential proteolytic activity pot

Expression of recombinant murine pregnancy-associated plasma
protein-A (PAPP-A) and a novel variant (PAPP-Ai) with differential
proteolytic activity
Rikke Søe
1
, Michael T. Overgaard
1
, Anni R. Thomsen
1
, Lisbeth S. Laursen
1
, Inger M. Olsen
1
,
Lars Sottrup-Jensen
1
, Jesper Haaning
1
, Linda C. Giudice
2
, Cheryl A. Conover
3
and Claus Oxvig
1
1
Department of Molecular and Structural Biology, Science Park, University of Aarhus, Denmark;
2
Department of Gynecology and
Obstetrics, Stanford University, Stanford, CA, USA;
3
Endocrine Research Unit, Mayo Clinic and Foundation, Rochester, USA

Murine pregnancy-associated plasma protein-A (PAPP-A)
cDNA encoding a 1545 amino-acid protein has been cloned.
We have also identified and cloned cDNA that encodes a
novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue
highly basic insert. The point of insertion corresponds to a
junction between two exons in the human PAPP-A gene.
The human intron flanked by these exons does not encode a
homologous corresponding insert, which is unique to the
mouse. The overall sequence identity between murine and
human PAPP-A is 91%, and murine PAPP-A contains
sequence motifs previously described in the sequence of
human PAPP-A. Through expression in mammalian cells,
we show that murine PAPP-A and PAPP-Ai are active
metalloproteinases, both capable of cleaving insulin-like
growth factor binding protein (IGFBP)-4 and -5. Cleavage
of IGFBP-4 is dramatically enhanced by the addition of
IGF, whereas cleavage of IGFBP-5 is slightly inhibited by
IGF, as previously established with human PAPP-A. Sur-
prisingly, however, quantitative analyses demonstrate that
the murine PAPP-Ai cleaves IGFBP-4 very slowly com-
pared to PAPP-A, even though its ability to cleave IGFBP-5
is unaffected by the presence of the insert. By RT-PCR
analysis, we find that both variants are expressed in several
tissues. The level of mRNA in the murine placenta does not
exceed the levels of other tissues analyzed. Furthermore, the
IGFBP-4-proteolytic activity of murine pregnancy serum is
not elevated. This is in striking contrast to the increase seen
in human pregnancy serum, and the expression of PAPP-A
in the human placenta, which exceeds other tissues at least
250-fold. Interestingly, the position of the insert of PAPP-Ai,

within the proteolytic domain, lies in close proximity to the
cysteine residue, which in human PAPP-A forms a disulfide
bond with the proform of eosinophil major basic protein
(proMBP). ProMBP functions as a proteinase inhibitor in
the PAPP-A–proMBP complex, but whether any mechan-
istic parallel on regulation of proteolytic activity can be
drawn between the insert of PAPP-Ai and the linkage to
proMBP is not known. Importantly, these data support the
development of the mouse as a model organism for the study
of PAPP-A, which must take into account the differences
between the mouse and the human.
Keywords: metalloproteinase; metzincin; insulin-like growth
factors; IGF binding proteins; pregnancy proteins.
Insulin-like growth factors (IGF)-I and -II are established
regulators of growth in many systems [1]. Their activity is
modulated by IGF binding proteins (IGFBPs), six of which
are known [2,3]. The IGFBPs bind IGF-I and -II with high
affinities, but proteolytic cleavage in the central region of an
IGFBP causes loss of its affinity for IGF. Thus, proteolysis
can be a prerequisite for the exertion of IGF activities [4].
Human pregnancy-associated plasma protein-A (PAPP-A)
was recently identified as a proteinase specific for IGFBP-4
[5] and IGFBP-5 [6]. Interestingly, its cleavage of IGFBP-4
is dramatically enhanced by the presence of IGF, whereas
the cleavage of IGFBP-5 is slightly reduced [6].
PAPP-A is a glycoprotein of 1547 residues [7], originally
isolated from the serum of pregnant women, but recently
also described in a number of human systems and shown to
be secreted from fibroblasts [5], osteoblasts [5,8], vascular
smooth muscle cells [9,10], and ovarian granulosa cells

[11,12]. In pregnancy, PAPP-A is synthesized in the human
placenta [13]. It reaches high levels in third trimester serum
( 50 mgÆL
)1
) [14], where it circulates as a disulfide bound
2 : 2 complex of 500 kDa with the proform of eosinophil
major basic protein (proMBP) [15,16]. The mature form of
the 206-residue proMBP, the 117-residue MBP, has a
calculated isoelectric point of 11 and is thus extremely basic.
MBP is cytotoxic and is found in granules of the eosinophil
leukocyte, from which it is secreted as a defense mechanism
of the immune system [17]. It has recently been demonstra-
ted that in the complex with PAPP-A, proMBP functions as
a proteinase inhibitor of unknown mechanism [18]. How-
ever, human pregnancy serum does show proteolytic
activity against IGFBP-4, because the amount of circulating
Correspondence to C. Oxvig, Department of Molecular and Structural
Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C,
DK-8000 Aarhus C, Denmark.
Fax: + 45 86123178, E-mail:
Abbreviations: PAPP-A, pregnancy-associated plasma protein-A;
PAPP-Ai, variant of PAPP-A with 29-residue insert in the proteolytic
domain; IGF, insulin-like growth factor; IGFBP, insulin-like growth
factor binding protein.
Enzyme: pregnancy-associated plasma protein-A (EC 3.4.24 ).
(Received 23 November 2001, revised 11 March 2002,
accepted 15 March 2002)
Eur. J. Biochem. 269, 2247–2256 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02883.x
PAPP-A is high (compared to PAPP-A levels elsewhere),
and because a minor fraction (< 1%) is found as a

noncomplexed PAPP-A dimer of about 400 kDa [18].
PAPP-A belongs to the metzincin superfamily of metal-
loproteinases [19,20], a diverse group of zinc peptidases
comprised of five families: the astacins (e.g. bone morpho-
genetic protein-1), the reprolysins or adamalysins (snake
venom proteinases, ADAMs), the serralysins (bacterial
proteinases), the matrix metalloproteinases (MMPs or
matrixins), and the pappalysins [21]. In addition to PAPP-
A, the latter includes PAPP-A2, a recently discovered
human homologue of PAPP-A showing 45% sequence
identity to PAPP-A [22]. All metzincins contain an elonga-
ted zinc-binding motif (HEXXHXXGXXH), which
coordinates the catalytic zinc ion of the active site. In
addition, they have a strictly conserved Met-residue, located
in the sequences at a variable distance (7–63 residues) to the
zinc-binding site [21], but in an invariable so-called Met-turn
in the three-dimensional structures [20]. PAPP-A and
PAPP-A2 further contain three lin-notch motifs (LNR1-3)
and five short consensus repeats (SCR1-5) [21].
Proteolysis of IGFBP-4 or -5 has been reported in
conditioned media from cultures of rat B104 neuroblastoma
cells [23], murine osteoblasts [24–26], rat ovarian granulosa
cells [27], and rat vascular smooth muscle cells [28].
However, whether PAPP-A exists in mouse as an active
enzyme is unknown.
We have cloned the cDNAs encoding murine PAPP-A
and a novel variant, PAPP-Ai, not known in humans, and
we have shown that mRNAs encoding both species are
expressed in several murine tissues. Recombinant expression
in mammalian cells allowed biochemical characterization of

murine PAPP-A and PAPP-Ai. Development of the mouse
as a model organism for the study of PAPP-A is an
important goal for understanding PAPP-A as a unique
metalloproteinase.
EXPERIMENTAL PROCEDURES
Cloning of cDNAs encoding murine PAPP-A and PAPP-Ai
Overlapping, murine cDNA clones encoding murine PAPP-
A of 1545 residues and a 1574-residue variant, PAPP-Ai,
were isolated using standard procedures. Nucleotides 1–3 of
the deposited sequences (AF439513, PAPP-A and
AF439514, PAPP-Ai) encode residue 1 (Fig. 1). Nucleotide
numbers below refer to the cDNA sequence of AF439513,
and residue numbers refer to its translated amino acid
sequence (Fig. 1), unless otherwise specified. In brief, a
murine cDNA library constructed in the lambda ZAP-
CMV vector (Stratagene) from murine placentas of a
17-day-pregnant mouse was screened using a
32
P-labeled
human PAPP-A cDNA probe derived from pPA1 [7]. From
a total of > 500 000 plaques, only two clones were found to
contain murine PAPP-A cDNA. Following rescue from the
lambda vector, the two clones were contained in the pBK-
CMV phagemid vector. One clone (E11) contained nucleo-
tides 1493–4638 of the murine PAPP-A cDNA sequence,
coding for residues 499–1545 of murine PAPP-A, followed
by a stop codon, and a 3¢-UTR sequence of  400
nucleotides. The sequence of the other clone was contained
within E11. No further sequence was obtained in subse-
quent rounds of re-hybridization using probes derived from

E11.
Using two rounds of RT-PCR with SuperTaq DNA
polymerase (HT Biotechnologies), the remaining nucleo-
tides of the murine PAPP-A cDNA sequence were obtained.
In the first round, cDNA was synthesized from term
placental RNA using a primer derived from E11 (nucleo-
tides 1695–1708). PCR was carried out with the 3¢ primer
derived from E11 (nucleotides 1664–1682), and the 5¢ primer
derived from the human PAPP-A cDNA sequence (nucle-
otides 3939–3959 of NM_002581). The resulting PCR
product was cloned into pCR 2.1-TOPO (Invitrogen). The
clone F2 contained nucleotides 478–1682, encoding residues
161–560. A variant, clone F2i, contained the same sequence,
in addition to an in-frame insert of 87 nucleotides (between
nucleotides 1232 and 1233 of AF439513, corresponding to
an insertion between amino acid residues 411 and 412). The
PAPP-A variant carrying this insert is denoted PAPP-Ai.
Similarly, clone F1 was obtained using an RT-primer
derived from F2 (nucleotides 847–866), a 3¢ primer derived
from F2 (nucleotides 507–526), and a 5¢ primer derived from
the human cDNA sequence (nucleotides 3438–3453 of
NM_002581). F1 contained nucleotides 1–525, encoding
residues 1–175. Several independent clones (of F2, F1, and
F2i) resulting from this PCR-based procedure were isolated
and found to be identical.
Generation of expression constructs
Expression constructs encoding full-length murine PAPP-A
and PAPP-Ai were made using a signal peptide previously
used for the expression of human PAPP-A [18]. First, T1218
of F2 was substituted with a G by overlap extension PCR

[29], using outer primers derived from the vector (nucleo-
tides 212–233 and 404–426 of pCR 2.1 TOPO), and
overlapping, inner primers derived from the PAPP-A
cDNA (nucleotides 1209–1232 and 1205–1226). This cre-
ated a silent BspEI site at residue Pro406 that will facilitate
future mutagenesis. The resulting PCR product was diges-
ted with PstI (nucleotide 1502) and XbaI (derived from the
vector), ligated into the PstI–ClaI fragment (nucleotides
1502–1845) of E11, and cloned into the XbaI/ClaIsitesof
pBluescript II SK+ (Stratagene) to obtain pB-F2PC (con-
taining nucleotides 478–1845 encoding residues 161–615 of
mPAPP-A).
Using the human PAPP-A expression construct
(pcDNA3.1-PAPP-A [18]) as a template and primers
derived from pcDNA3.1+ (Invitrogen) (nucleotides 792–
812) and pcDNA3.1-PAPP-A/F1 (5¢-GCCCCTCGCCGC
GCTCGAGGCCG-3¢), a PCR product containing a
HindIII site followed by nucleotides encoding the signal
peptide (MKDSCITVMAMALLSGFFFFAPASS) and
murine PAPP-A residues 1–4 was obtained. Using F1 as a
template, a primer derived in part from nucleotides 1–13
(5¢-GGCCTCGAGCGCGGCGAGGGGCG-3¢), and a
primer derived from the vector (nucleotides 212–233 of
pCR 2.1 TOPO), an overlapping PCR product was
generated. The two PCR products were then used in an
overlap reaction, and the resulting product cloned into
pCR-Blunt II-TOPO (Invitrogen) to obtain pCR-sp F1
(encoding the signal peptide and residues 1–175 of
mPAPP-A).
2248 R. Søe et al. (Eur. J. Biochem. 269) Ó FEBS 2002

Next, using pB-F2PC and pCR-sp F1 as templates,
outer primers derived from the vector sequences (nucleo-
tides 625–645 of pBluescript II SK+ and nucleotides 792–
812 of pcDNA3.1+ (Invitrogen), and inner primers derived
from the murine PAPP-A cDNA sequence (nucleotides
478–499 and 504–524), a PCR product encoding the signal
peptide and residues 1–615 was generated and cloned into
pCR-Blunt II-TOPO. Finally, the HindIII–ClaIfragment
was excised from this construct, ligated to the ClaI–BamHI
fragment (encoding residues 616–1545) of E11, and cloned
into pcDNA3.1+, to finally obtain pcDNA3.1-mPA. Using
F2i rather than F2, pBF2iPC, and further pcDNA3.1-
Fig. 1. Alignment of the murine (mPA, 1545 residues) and human (hPA, 1547 residues) PAPP-A sequences. A variant of the murine protein, PAPP-
Ai, carries a 29-residue, basic insert whose amino acid sequence and position within the proteolytic domain (between residues 411 and 412) is
emphasized. The extent in primary structure of the proteolytic domain, as recently defined [21], is indicated by the shading of residues 270–581. The
human sequence [7] (GenBank accession number X68280) is shown only where different from the murine sequence. Murine PAPP-A contains
91.1% residues which are also found in the human protein; all of the 82 cysteine residues are conserved. The elongated zinc-binding site (residues
480–490) and the Met-turn residues (552–556) [21] are shown in bold and underlined. Other defined stretches of amino acids are three lin-notch
motifs (LNR1-3) and five short consensus repeats (SCR1-5). The PAPP-A cysteine residue known to be engaged in disulfide bonding to proMBP in
the human PAPP-A–proMBP complex, the human Cys381 [15], is pointed out.
Ó FEBS 2002 Murine PAPP-A and PAPP-Ai (Eur. J. Biochem. 269) 2249
mPAi, was generated in parallel by the same procedure. All
PCRs were carried out using Pfu DNA polymerase
(Stratagene), and the final constructs were verified by
sequence analysis.
Tissue culture and expression of recombinant proteins
Human embryonic kidney 293T cells (293tsA1609neo) [30]
were maintained in high-glucose Dulbecco’s modifies
Eagle’s medium supplemented with 10% fetal bovine
serum, 2 m

M
glutamine, nonessential amino acids, and
gentamicin (Life Technologies). Cells were plated onto 6-cm
tissue culture dishes, and were transfected 18 h later by
calcium phosphate coprecipitation [31] using 10 lgof
plasmid DNA prepared by QIAprep Spin Kit (Qiagen).
After a further 48 h, the supernatants were harvested and
cleared by centrifugation. Expression plasmids containing
murine PAPP-A, murine PAPP-Ai, or human PAPP-A [18]
were used for transfection. The level of human PAPP-A in
culture supernatants, determined by ELISA specific for
human PAPP-A, was about 5 lgÆmL
)1
or 25 n
M
(of
200 kDa PAPP-A monomer), as reported previously [18].
No immunoassay is available for murine PAPP-A, but
based on quantitative comparison (cf. below) of the activity
against both IGFBP-4 and IGFBP-5 (not shown), it can be
suggested that supernatant from cells transfected murine
PAPP-A cDNA also contain  25 n
M
. However, superna-
tant from cells transfected with murine PAPP-Ai cDNA
showed very little activity against IGFBP-4 and less activity
against IGFBP-5, compared to murine PAPP-A. The levels
of murine PAPP-A and PAPP-Ai were therefore compared
by Western blotting using polyclonal rabbit antibodies
raised against human PAPP-A/proMBP complex [16], and

enhanced chemiluminescence (ECL Plus, Amersham).
Although raised against human protein, this preparation
of antibodies did result in a signal in Western blotting
experiments. A threefold difference in expression levels was
found (not shown) that was subsequently adjusted for.
Expression of recombinant binding proteins was similarly
carried out, and purification was performed as recently
described for IGFBP-4 [21] and IGFBP-5 [22].
Measurement of proteolytic activity against IGFBP-4
and -5
All digests were carried out in 100 m
M
NaCl, 1 m
M
CaCl
2
,
50 m
M
Tris, pH 7.5 using purified and iodinated IGFBP-4
[21] and IGFBP-5 [22]. The reaction mixtures were analyzed
by nonreducing SDS/PAGE (16%) followed by autoradi-
ography. The material loaded per lane (10 lL) contained
25 000 c.p.m. ( 2.5 ng or 7 n
M
) of radiolabeled binding
protein. All reactions were incubated at 37 °C (up to 72 h)
as specified in the text. Both of the binding proteins were
expressed as C-terminally tagged proteins causing the
PAPP-A cleavage products to comigrate, as detailed

previously [6].
For qualitative assays (Fig. 2), unlabeled, purified
IGFBP-4 or -5 was added to a final concentration of
30 n
M
. Standard serum containing media from cells trans-
fected with empty vector or PAPP-A/PAPP-Ai cDNA were
used ( 0.1 n
M
proteinase), and 40 n
M
IGF-II (Bachem)
was added to some reactions as specified. Quantitative
assays (Fig. 4A,B), recently developed [6], were carried out
after addition of 144 /130 n
M
unlabeled IGFBP-4/)5,
167 n
M
IGF-II, and equal amounts of proteinase (0.1 n
M
)
contained in culture supernatants. For each experiment, one
reaction of 120 lL was set up, from which samples of 10 lL
were taken out at selected time points, stopped by the
addition of 5 m
M
EDTA, and frozen. Following SDS/
PAGE, the degree of cleavage was determined by measuring
band intensities with a

PHOSPHORIMAGER
(Molecular
Dynamics) [6,21]. The background signal from a control
reaction using medium from mock-transfected cells was
subtracted, and the degree of cleavage was plotted as a
function of time. For evaluation of IGFBP-4 proteolytic
activity in sera, blood was drawn from nonpregnant and
pregnant (18 days) mice, and from nonpregnant and
pregnant women (at term). Serum (0.5 lL) was used in
each reaction along with 40 n
M
of added IGF-II.
Analysis of tissue expression by RT-PCR
Selected tissues from nonpregnant and pregnant mice were
frozen in liquid nitrogen. Individual tissues (approximately
30 mg) was homogenized, further processed using QIA-
shredder (Qiagen), and RNA was prepared using RNeasy
Fig. 2. Proteolytic activity of recombinant murine PAPP-A and PAPP-
Ai against IGFBP-4 and -5. (A) Radiolabeled IGFBP-4 was incubated
(24 h) with medium from mock-transfected cells (lane 1), with medium
from cells transfected with murine PAPP-A cDNA (lanes 2–3), with
murine PAPP-Ai cDNA (lanes 4–5), or with human PAPP-A cDNA
(lane 6). Below each lane the absence (–) or presence (+) of 40 n
M
added IGF-II is indicated. B: Similar experiment carried out with
radiolabeled IGFBP-5 (except IGF-II was not added in lane 6, as
indicated). The positions of molecular mass markers, and the positions
of intact and cleaved IGFBP-4 and -5 are indicated. The C-terminal
tag on both of the binding protein causes their PAPP-A cleavage
products to comigrate, and thus appear as one band, as detailed pre-

viously [6].
2250 R. Søe et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Mini Kit (Qiagen). First strand cDNA was synthesized with
Thermoscript RT-PCR System (Life Technologies) and a
primer derived from the murine PAPP-A sequence (nucleo-
tides 1425–1448 of AF439513). PCR primers were chosen
that would amplify a nucleotide stretch spanning the cDNA
encoding the 29-residue insert of PAPP-Ai (nucleotides
1117–1137 and nucleotides 1275–1293 of AF439513),
resulting in a 177-bp product specific for cDNA derived
from PAPP-A mRNA, and 264-bp product specific for
PAPP-Ai mRNA. To analyze for the presence of a
corresponding insertion of human PAPP-A, the same
analysis was performed on human tissue using an RT
primer (nucleotides 4885–4908 of NM_002581) and two
equivalent PCR primers (nucleotides 4577–4597 and nucleo-
tides 4735–4753 of NM_002581) derived from the human
PAPP-A sequence. Reactions (33 cycles of PCR) were
carried out using SuperTaq DNA polymerase (HT Bio-
technologies).
RESULTS
Isolation of cDNAs encoding murine PAPP-A
and PAPP-Ai
A cDNA library prepared from murine placenta was
screened with a nucleotide probe encoding human PAPP-A.
Only two clones, covering the C-terminal two thirds of the
sequence of murine PAPP-A, were found by hybridization.
The remaining sequence was obtained by an RT-PCR based
procedure, as detailed above.
The deduced amino-acid sequence of murine PAPP-A

contains 1545 residues, 137 of which differ from human
PAPP-A. Thus, PAPP-A is highly conserved with 91.1%
identical residues between the two species (Fig. 1). Extended
stretches of identical residues occur, but positions that
deviate appear evenly distributed. Importantly, all of the 82
cysteine residues are conserved.
Of particular interest, we have demonstrated the existence
of an mRNA species encoding a variant of PAPP-A with 29
residues (QSIRKRAHVVEESWLPHGKQKAKKRKR
TR) inserted in the proteolytic domain, between Arg411
and Ala412 (Fig. 1). We denoted this variant PAPP-Ai. The
point of insertion does not interrupt any of the predicted
secondary-structure elements of PAPP-A, but is located
next to the N-terminal end of the pappalysin-specific a helix
H-ii, between the canonical b strands S3 and S4 of the
metzincins [21]. With 11 basic (six Lys and five Arg) and two
acidic (both Glu) residues, the inserted stretch is highly
basic. The nucleotide sequence encoding murine PAPP-A
has been deposited in the GenBank database under the
accession number AF439513, and the sequence encoding
PAPP-Ai under the accession number AF439514.
Does the human PAPP-A gene encode a similar insert?
The relevant portion of the human PAPP-A amino-acid
sequence is encoded by the genomic sequence of GenBank
accession number AB020878: nucleotides 35 894–36 958
and 56 217–56 362 encode human PAPP-A residues 59–413
and 414–461 (corresponding to murine residues 57–411 and
412–459, see Fig. 1). Thus, the point of insertion of the 29-
residue insert of murine PAPP-Ai corresponds to a junction
between two exons of the human gene, and the nucleotides

36.959–56.216 (of AB020878) either (a) correspond to a
single intron of the human gene, or (b) contain an exon
corresponding to the 29-residue murine insert. Within this
stretch of  20 000 nucleotides, no human sequence was
found that, when translated, showed significant sequence
similarity to the insert of murine PAPP-Ai. We further
looked for ORFs that were flanked by donor and acceptor
splice sites [32], and found five candidate stretches of 15–76
amino acids. None of these, however, matched the required
exon phase. In addition, none of them were represented in
the human subset of the GenBank expressed sequence tag
(EST) database, whereas > 100 human EST sequences
encoded a sequence spanning the exon/exon junction at
residues 413/414. Based on this, the presence of an insert at
this position is unique to the mouse. It has previously been
found [33], using a human cDNA probe, that the murine
genome contains only one PAPP-A gene. Thus, PAPP-A
and PAPP-Ai mRNA likely results from alternative splicing
of a transcript from the same gene.
Expression and functional analysis of murine PAPP-A
and PAPP-Ai
The proteolytic domain of murine PAPP-A, as recently
defined in the sequence of human PAPP-A [21] (Fig. 1),
does not deviate from the overall degree of conservation
(89.7% of the 312 residues are conserved). All residues of
the zinc binding consensus are conserved (Fig. 1), which
strongly suggests that the murine protein is also an active
metalloproteinase. To experimentally verify this, full-length
constructs encoding PAPP-A and PAPP-Ai were made,
cloned into a mammalian expression vector, and used for

transient transfection of 293T cells.
The presence of recombinant murine PAPP-A in culture
supernatants of transfected cells was then confirmed by the
detection of proteolytic activity against IGFBP-4 and
IGFBP-5 (Fig. 2). Medium from cells transfected with
empty vector did not have the ability to cleave IGFBP-4
(Fig. 2A, lane 1), but medium from cells transfected with
murine PAPP-A cDNA caused specific cleavage in the
presence of added IGF-II (Fig. 2A, lane 2). In the absence
of IGF-II, proteolysis was dramatically less pronounced
(Fig. 2A, lane 3). This highlights the enhancing effect of
IGF on proteolysis of IGFBP-4, which is widely recognized
for human PAPP-A [6]. PAPP-Ai also specifically cleaved
IGFBP-4 in an IGF-dependent manner (Fig. 2, lanes 4 and
5). Interestingly, however, the amount of proteolysis (in the
presence of IGF) appeared to be much lower when
compared to PAPP-A (Fig. 2A, lanes 2 and 4).
In a similar experiment, we found that both PAPP-A and
PAPP-Ai were able to specifically cleave IGFBP-5 inde-
pendent of IGF (Fig. 2B). In contrast to the proteolysis of
IGFBP-4, the presence of added IGF slightly hampered the
proteolysis of IGFBP-5, as recently demonstrated with
human PAPP-A [6].
To verify that both PAPP-A and PAPP-Ai are expressed
as full-length proteins, we performed Western blotting using
polyclonal antibodies against the human PAPP-A/proMBP
complex, which were found to recognize murine PAPP-A
and PAPP-Ai immobilized on a PVDF membrane. This
experiment demonstrates that both species are in fact
expressed as dimers of  400 kDa (Fig. 3), as human

PAPP-A.
Ó FEBS 2002 Murine PAPP-A and PAPP-Ai (Eur. J. Biochem. 269) 2251
The basic insert of PAPP-Ai restricts proteolysis
of IGFBP-4, but not IGFBP-5
Rates of proteolysis cannot be compared using a fixed time
of incubation, as in the experiment described above, where
samples were incubated for 24 h (Fig. 2). During this
incubation, only a fraction of IGFBP-4 had been degraded
by PAPP-Ai (Fig. 2, lane 4). In contrast, the almost
complete proteolysis by PAPP-A (Fig. 2, lane 2) may have
occurred in much less time.
To more accurately describe this apparent difference in
activity of PAPP-A and PAPP-Ai towards IGFBP-4,
analyses were carried out using a recently developed
quantitative assay [6]. Samples were removed from the
reactions at several different time points, and the degree of
cleavage was determined by SDS/PAGE followed by
measurement of band intensities with a
PHOSPHORIMAGER
.
This revealed that in comparison with PAPP-A, PAPP-Ai is
much less proteolytically active against IGFBP-4. As
measured after 180 min of incubation, the activity of
PAPP-Ai is only about 10% of the activity of PAPP-A
(Fig. 4A).
A similar time course experiment was performed with
IGFBP-5 as the substrate. Surprisingly, PAPP-A and
PAPP-Ai degraded IGFBP-5 with very similar rates
(Fig. 4B). We therefore conclude that the basic insert of
29 residues carried by PAPP-Ai differentially affects its

substrate specificity; PAPP-A proteolysis of IGFBP-5 is not
affected by the presence of the insert, but the ability of
PAPP-A to cleave IGFBP-4 is dramatically reduced.
mRNA species encoding both PAPP-A variants
are present in several tissues
To verify the existence of both PAPP-A and PAPP-Ai
mRNA in murine tissues, RT-PCR analysis was carried out
using PCR primers spanning the site of insertion in the
nucleotide sequence. Most of the tissues analyzed contained
both mRNA species; in general, PAPP-A mRNA appeared
to be the most abundant (Fig. 5A). Of particular interest,
although the method does not allow quantitative compar-
isons between tissues, expression of PAPP-A and PAPP-Ai
mRNA in the murine placenta appeared similar to levels in
other tissues analyzed. The expression of PAPP-A mRNA
in the human placenta, in contrast, exceeds expression in
other human tissue by > 250-fold [34].
To experimentally verify the absence of a human
transcript encoding an insert between residues 413 and
414, RT-PCR with primers derived from the corresponding
part of the human PAPP-A sequence was also carried out
using cDNA derived from human placenta as a template
(Fig. 5B). No band of increased size was seen, providing
Fig. 4. Degradation of IGFBP-4 and IGFBP-5 by murine PAPP-A and
PAPP-Ai as a function of time. Recombinant murine PAPP-A (s and
PAPP-Ai (d) (both at 0.1 n
M
) were incubated with radiolabeled
IGFBP-4 (144 n
M

)(A)orIGFBP-5(130n
M
) (B) in the presence of
added molar excess of IGF-II. Samples of the reaction mixtures were
taken at various time points, and the degree of cleavage was determined
by densiometry using a PhosphorImager after separation by SDS/
PAGE. Values are average of three independent experiments ± SD.
Fig. 3. Western blotting of murine PAPP-A and PAPP-Ai. Culture
supernatants from cells transfected with murine PAPP-A cDNA
(lane 1), empty vector (lane 2), or PAPP-Ai cDNA (lane 3) were
separated by nonreducing SDS/PAGE and blotted onto a
poly(vinylidene flouride) membrane for immunodetection. Polyclonal
antibodies against human PAPP-A/proMBP were found to be effective
in this procedure, thus recognizing the denatured murine PAPP-A and
PAPP-Ai. Positions of molecular mass markers are indicated.
2252 R. Søe et al. (Eur. J. Biochem. 269) Ó FEBS 2002
further evidence for the lack of a human counterpart of the
murine PAPP-Ai variant.
Murine pregnancy serum does not contain proteolytic
activity against IGFBP-4
We finally looked for IGFBP-4 proteolytic activity in
murine pregnancy serum (Fig. 6). Even under conditions of
prolonged incubation, neither nonpregnant (Fig. 6, lanes
1–2) nor pregnant (Fig. 6, lanes 3–4) murine serum showed
any ability to convert IGFBP-4 into fragments character-
istic of PAPP-A proteolysis. However, two very faint
different bands seen with nonpregnant serum, but not
pregnant serum, indicated the possible presence in non-
pregnant murine serum of a different proteinase with very
limited ability to cleave IGFBP-4. Human nonpregnancy

serum did not show any cleavage of IGFBP-4 (Fig. 6,
lane 5), but human pregnancy serum showed the expected
cleavage caused by PAPP-A (Fig. 6, lane 6–7).
To exclude the possibility that the lack of PAPP-A
activity in murine pregnancy serum was caused by an
unknown inhibitor, we compared proteolysis of IGFBP-4
by recombinant murine PAPP-A in the absence and in the
presence of added murine pregnancy serum (not shown).
No difference in activity was seen, supporting the conclusion
that murine pregnancy serum does not contain PAPP-A.
DISCUSSION
We have cloned a cDNA encoding murine PAPP-A of 1545
residues, and we have identified a cDNA encoding a variant,
PAPP-Ai, in which 29 residues are inserted in the proteolytic
domain. Through expression in mammalian cells, we show
that both PAPP-A and PAPP-Ai are active proteinases of
about 400 kDa. Further analyses demonstrate that (1) both
PAPP-A and PAPP-Ai cleave IGFBP-4 in an IGF
dependent manner, but that PAPP-Ai is a much slower
IGFBP-4 proteinase than PAPP-A (2) in contrast, both
PAPP-A and PAPP-Ai cleave IGFBP-5 independent of
IGF at very similar rates (3) mRNA encoding PAPP-A and
PAPP-Ai are both present in most murine tissues analyzed,
and (4) murine pregnancy serum does not possess an
elevated level of proteolytic activity against IGFBP-4, in
striking contrast to human pregnancy serum.
As PAPP-A is abundantly expressed in the human
placenta [13,34], we unexpectedly found only two partial
PAPP-A cDNA clones upon hybridization with human
cDNA to a murine placental cDNA library. The remaining

sequence (the N-ternimal 498 residues) was obtained by a
PCR procedure using specifically primed placental cDNA
as the template. Of the 1545 residues of murine PAPP-A,
91.1% are also found in human PAPP-A. The two
sequences can be aligned without introducing gaps, except
for missing residues at two single positions (corresponding
to human residues 6 and 27). Critical residues, such as the 82
cysteine residues, residues of the elongated zinc-binding
consensus, and the Met-turn residues, are conserved
between mouse and man (Fig. 1). Searching the GenBank
database revealed 8 murine EST sequences derived from
PAPP-A mRNA (none of these originate from placenta),
and two partial cDNA sequences (AF260433 and
AF258461) encoding murine PAPP-A, but lacking  500
nucleotides corresponding to residues 1 though 178.
Because of the limited number of available matching EST
clones, RT-PCR was performed on a series of murine tissues
(Fig. 5A). A set of primers was selected that tested for the
Fig. 6. Comparison of IGFBP-4 proteolytic activity in murine and
human pregnancy serum. Radiolabeled IGFBP-4 was incubated (72 h)
with serum from nonpregnant female mice (lanes 1–2), serum from near
term (18 days) pregnant mice (lanes 3–4), serum from a nonpregnant
woman (lane 5), and two different samples of term human pregnancy
serum (lanes 6–7). All reactions were in the presence of added IGF–II.
The positions of intact and cleaved IGFBP-4 are indicated.
Fig. 5. RT-PCR analysis of murine PAPP-A and PAPP-Ai mRNA.
(A) A panel of cDNA preparations derived from murine tissues was
screened by PCR for the absence or presence of mRNA encoding
PAPP-A and PAPP-Ai, respectively. The presence of both mRNA
species in several of the tissues analyzed is evidenucleotides Individual

tissues tested and bands of 177 and 264 bp, corresponding to PAPP-A
and PAPP-Ai mRNA, respectively, are indicated. (B) A similar
experiment using equivalent primers derived from the human PAPP-A
cDNA sequence and template derived from human placenta. No band
corresponding to the murine 264 bp band was observed. For com-
parison, the PCR products obtained with murine PAPP-A and PAPP-
Ai cDNA are also shown in the lanes labeled Ômurine PAPP-AÕ and
Ômurine PAPP-AiÕ.
Ó FEBS 2002 Murine PAPP-A and PAPP-Ai (Eur. J. Biochem. 269) 2253
presence of both PAPP-A and PAPP-Ai mRNA at the same
time. Most of the tissues analyzed were found to contain
both species. Although the assay used is not quantitative, it
is fair to conclude that expression in the murine placenta
does not differ dramatically from other tissues analyzed.
This is in accordance with the above findings, but in striking
contrast to semiquantitative analyses of PAPP-A mRNA
expression in human tissues, which revealed that expression
in the human placenta exceeds expression in other tissue
250- to 3000-fold [34]. In the human placenta, PAPP-A
mRNA is abundantly synthesized in the syncytiotropho-
blast [13], the chorionic epithelium of fetal origin which is in
direct contact with the maternal blood. Based on this direct
contact, the placenta of man (and other primates) and the
placenta of mouse (and other rodents) are classified together
as hemochorial. In contrast, the placentas of horses, pigs,
ruminants, cats and dogs etc. are of different types with
more separating layers. Thus, most likely, the synthesis of
PAPP-A does not correlate with placental type. The
detected PAPP-A mRNA of the murine placenta may
originate from cells of fetal or maternal connective tissue.

The relatively high levels of PAPP-A circulating in
human pregnancy serum most likely originate from the
placenta. As PAPP-A mRNA expression in the murine
placenta is not elevated compared to other tissues, we did
not expect to find elevated levels of IGFBP-4 proteolytic
activity in late mouse pregnancy serum, which was
confirmed (Fig. 6). Previously, the presence of intact
IGFBP-4 (identified as a band of 24 kDa by Western
ligand blotting) in murine late pregnancy serum provided
indirect evidence that an IGFBP-4 proteinase was absent
from the circulation of mouse [35], although human
pregnancy serum contained detectable intact IGFBP-4 only
before gestational week 10 [36]. The lack of IGFBP-4-
specific proteolysis in murine pregnancy serum, as found
here, is thus in line with the earlier findings, but it could not
be ruled out previously that the apparently constant level of
intact IGFBP-4 in murine pregnancy was caused by an
increase in synthesis along with increased proteolysis.
The presence of PAPP-A mRNA in all murine tissues
analyzed parallels the ubiquitous occurrence of PAPP-A in
human tissues. Several recent papers have reported proteo-
lytic activity against IGFBP-4 or -5 in conditioned media
from cultures of mouse or rat cells [23–28]. Hence, PAPP-A
and PAPP-Ai are obvious candidate proteinases in these
systems. However, a murine homologue of PAPP-A2 may
also be responsible in part for proteolysis in these different
systems. Human PAPP-A2 was recently identified and
demonstrated to cleave IGFBP-5 [22]. By searching the
GenBank database for murine nucleotide sequences enco-
ding protein similar to human PAPP-A2, we determined the

existence of this protein in mouse. Interestingly, the found
PAPP-A2 sequence stretches (AK005504, BB462397, and
AI157031, for example), showed a lower degree of conser-
vation (64–83% in stretches of 78–120 residues) than the
91% observed between human and murine PAPP-A.
Our cloning of cDNA encoding both PAPP-A and
PAPP-Ai allowed expression in mammalian cells and
functional analyses of the recombinant proteins. Of partic-
ular interest is the finding that PAPP-Ai does not readily
cleave IGFBP-4, and that, in contrast, PAPP-A and PAPP-
Ai cleave IGFBP-5 with very similar rates. This immediately
suggests that proteolysis of IGFBP-4 might be regulated by
the control of PAPP-A/PAPP-Ai mRNA splicing. Both
mRNA species are present in all murine tissues analyzed.
However, at the level of individual cells or cell types within
the tissues, PAPP-A and PAPP-Ai mRNA may be differ-
entially expressed.
Sequence stretches similar to the 29-residue insert
sequence was not found within the genomic sequence of
human PAPP-A that potentially encodes a corresponding
human insert. But a functional role of the insert of the
murine PAPP-Ai is strongly suggested from the above
experiments, even though the mechanism of its action
cannot be predicted. Curiously, the site of insertion within
the proteolytic domain of PAPP-A lies in close proximity to
the cysteine residue which in the human PAPP-A/proMBP
complex forms a disulfide bond to proMBP [15] (see Fig. 1).
As mentioned above, proMBP functions as a proteinase
inhibitor in the PAPP-A/proMBP complex [18], but whe-
ther any mechanistic parallel on regulation of proteolytic

activity can be drawn between the insert of PAPP-Ai and
the linkage to proMBP is not known. A striking common
feature of the 29-residue insert and MBP (of 117 residues) is
their pronounced basic characters, which may be important
for the basis of their actions.
Even though the pregnancy protein PAPP-A is practi-
cally absent from the murine placenta, the mouse may prove
useful for the study of physiological roles of PAPP-A
outside this tissue. Development of the mouse as a model for
the study of PAPP-A must take into account the existence of
PAPP-Ai. In contrast to the human system, specific
proteolytic activity against IGFBP-4 will depend on whe-
ther PAPP-A or PAPP-Ai functions in a given system.
Importantly, the availability of an expression system for
recombinant murine PAPP-A will allow generation anti-
bodies against murine PAPP-A, highly desired for efficient
use of a murine model, as well as detailed mapping of
monoclonal antibodies by homology substitution. Further,
murine PAPP-A is now available for biochemical studies of
the fifth metzincin family, the pappalysins.
ACKNOWLEDGEMENTS
This work was supported by grants from the Danish Medical Research
Council, the Alfred Benzon Foundation, the Novo Nordic Foundation,
and the National Institute of Health (HD31579-07).
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