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: co@mbio.aau.dk 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). REFERENCES 1. Daughaday, W.H. & Rotwein, P. (1989) Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocrinol. Rev. 10, 68–91. 2. Hwa, V., Oh, Y. & Rosenfeld, R.G. 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We have also identified and cloned cDNA that encodes a novel variant of PAPP -A, PAPP-Ai,. 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