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

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

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

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

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

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

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-

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

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

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

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

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.

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

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