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J. Biol. Chem., Vol. 276, Issue 24, 21849-21853, June 15, 2001
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From the
Received for publication, October 19, 2000, and in revised form, March 12, 2001
A novel metalloproteinase with similarity
to pregnancy-associated plasma protein-A (PAPP-A), which we denoted
PAPP-A2, has been identified. Through expression in mammalian cells we
showed that recombinant PAPP-A2 polypeptide of 1558 residues
resulted from processing of a 1791-residue prepro-protein. Unlike
PAPP-A, PAPP-A2 migrated as a monomer (of 220 kDa) in non-reducing
SDS-polyacrylamide gel electrophoresis. The prepro-parts of PAPP-A2 and
PAPP-A are not homologous, but mature PAPP-A2 shares 45% of its
residues with PAPP-A. Because PAPP-A specifically cleaves insulin-like growth factor-binding protein (IGFBP)-4, one of six known modulators of IGF-I and -II, we looked for a possible PAPP-A2 substrate among the
members of this family. We showed that PAPP-A2 specifically cleaved
IGFBP-5 at one site, between Ser-143 and Lys-144. In contrast to the
cleavage of IGFBP-4 by PAPP-A that strictly requires the presence of
IGF, the cleavage of IGFBP-5 by PAPP-A2 was IGF-independent. Recent
data firmly establish PAPP-A and IGFBP-4 as an important functional
pair in several systems. Because of its close relationship with PAPP-A,
both structurally and functionally, PAPP-A2 is a likely candidate
IGFBP-5 proteinase in many tissues and conditioned media where IGFBP-5
proteolysis has been reported.
Pregnancy-associated plasma protein-A
(PAPP-A)1 has recently been
shown to specifically cleave insulin-like growth factor-binding protein-4 (IGFBP-4) (1), one of six modulators of IGF-I and -II
activity (2). Cleavage of IGFBP-4 causes release of bound IGF that in
turn interacts with its cellular receptor (3). Interestingly, the
cleavage of IGFBP-4 by PAPP-A strictly depends on the presence of IGF
(1). It has been established that PAPP-A is the IGFBP-4 proteinase
secreted from fibroblasts (1), osteoblasts (1, 4), marrow stromal cells
(1), and vascular smooth muscle cells (5) and is present in pregnancy
serum (6) and ovarian follicular fluid (7).
PreproPAPP-A is synthesized as a 1627-residue protein, which is
processed into mature PAPP-A of 1547 residues (8, 9). In most systems,
PAPP-A appears to exist as a homodimer of 400 kDa (1). However, in
pregnancy serum, and possibly elsewhere, >99% of PAPP-A is found as a
disulfide bound 2:2 complex with the highly and unusually glycosylated
proform of eosinophil major basic protein (proMBP) (10-12). In this
covalent complex, proMBP functions as an inhibitor of the proteolytic
activity of PAPP-A (6).
No proteins with global homology to PAPP-A have been described. Here we
report the identification of a novel protein, PAPP-A2, which is a
homologue of PAPP-A. Expression of recombinant PAPP-A2 allowed us to
establish that PAPP-A2 is an active metalloproteinase, that it
specifically cleaves IGFBP-5, and that it therefore likely functions
in the same growth regulatory system as PAPP-A.
Identification and Cloning of a Nucleotide Sequence Encoding
PAPP-A2
Searching (13) public databases for DNA sequences that when
translated showed homology to the preproPAPP-A protein
sequence2 revealed two
genomic clones, AL031734 and AL031290, containing coding sequence
stretches corresponding to ~25% of the N-terminal amino acid
sequence of PAPP-A (residues 96-493)2, and 15% of the
C-terminal (residues 1393-1593). Based on their co-localization on
chromosome 1 (1q24), we hypothesized the existence of a novel protein
similar to PAPP-A, which we named PAPP-A2. We first established a
coding cDNA sequence aligning with the ~60% remaining, central
region of PAPP-A; cDNA was synthesized using human placental
mRNA as a template and a primer derived from AL031290
(5'-GCTCACACACCACAGGAATG-3'). With primers derived from AL031734
(5'-GGCTGATGTGCGCAAGACCTG-3') and AL031290 (5'-GCATTGTATCTTCAGGAGCTTG-3'), a PCR product was generated that corresponded to the central region, a total of 908 amino acids (PAPP-A2
residues 665-1572). Manual inspection of the genomic sequence of
AL031734 revealed that the open reading frame of the sequence stretch
aligning with PAPP-A continued further in the 5' direction; nt
102646-103566 encode a polypeptide sequence of 307 residues that
starts with a methionine residue. Thus, the same cDNA
preparation was used to obtain a contiguous cDNA sequence encoding
the N-terminal portion of PAPP-A2 using primers from AL031734
(5'-GAAGTTGACTTCTGGTTCTGTAG-3') and from the central region
(5'-CCCTGGGAAGCGAGTGAAGCC-3').
A stop codon was present in the sequence of AL031290 corresponding to
PAPP-A residue 1618. Therefore, cDNA was synthesized using
placental mRNA and a primer originating from AL031290 further 3' to
this stop codon (5'-GCATTTCTTATAAGATCCTTCATGC-3'). A contiguous
cDNA corresponding to the C-terminal of PAPP-A2 was obtained in a
PCR with primers from the central region
(5'-GACAGCTGTCCGTCATTGCTGC-3') and from AL031290
(5'-CTTACTGCCTCTGAGGCAGTGG-3'). All PCRs were carried out with
Pfu polymerase (Stratagene). The three overlapping PAPP-A2
cDNA fragments obtained were cloned into the vector
pCR-BluntII-TOPO (Invitrogen), and referred to as p2N, p2Mid, and p2C,
respectively. The entire sequence of 5376 nucleotides encodes
preproPAPP-A2 of 1791 residues3.
Identification of EST Sequences
A cluster of EST sequences matching the genomic sequence of
AL031290 was identified around nt 64000-66000 of AL031290 starting
~1.2 kb from the 3' end of the PAPP-A2 encoding sequence. The
existence of mRNA connecting the coding region of PAPP-A2 and this
cluster was verified by PCR using primers from AL031290 (5'-GGAAAGAGCAGAGTTCACCCAT-3', nt 64900-64879 of AL031290), the
PAPP-A2 encoding sequence (5'-CCGTCTTAGTCCACTGCATCC-3', nt 20499-20519
of AL031290 and nt 5171-5191 of AF311940), and oligo(dT)-primed
placental cDNA as a template (12). As expected, the size of the
resulting product was 2.2 kb, further demonstrating the existence of a
PAPP-A2 mRNA with a 3' untranslated region of ~3 kb.
Plasmid Construction
pPA2, Encoding Wild-type PreproPAPP-A2--
The
NotI-BamHI fragment from p2C was cloned into
pBluescriptIISK+ (Stratagene) to obtain p2CBlue. The
NotI-SpeI fragment from p2N and the
SpeI-BclI fragment from p2Mid were ligated into
the NotI/BclI sites of p2CBlue to obtain
p2NMidCBlue containing the entire PAPP-A2 cDNA. The
NotI-ApaI fragment of pBluescriptIISK+ was
ligated into the NotI/ApaI sites of the mammalian
expression vector pcDNA3.1+ (Invitrogen) to obtain a modified
version of this vector, pcDNA-NA. The full-length cDNA was then
excised from p2NMidCBlue with NotI and XhoI and
cloned into pcDNA-NA to obtain pPA2.
pPA2-KO--
By overlap extension PCR (14), a construct encoding
an inactive variant of pPA2, pPA2-KO, was made in which Glu-734 is
replaced with a Gln (E734Q). Outer primers were
5'-CGCTCAGGGAAGGACAAGGG-3' (5' end primer, nt 976-995 of AF311940) and
5'-CTAGAAGGCACAGTCGAGGC-3' (3' end primer, nt 1040-1021, sequence of
vector pcDNA3.1+). Overlapping, mutated internal primers were
5'-TGTCCCACTTGATGGATCATGGTGTCGGTGTGG-3' and
5'-CCATGATCCATCAAGTGGGACATGTTCTGGGAC-3'. The mutated fragment was
digested with XbaI and XhoI and swapped into pPA2
to generate pPA2-KO.
pPA2-mH--
Two primers (5'-GAGGGCCTGTGGACCCAGGAG-3', nt
4906-4926 of AF311940, and 5'-GACGTAAAGCTTCTGATTTTCTTCTGCCTTGG-3', nt
5373-5354 of AF311940, preceded by a HindIII site, AAGCTT)
were used in a PCR with pPA2 as the template to generate a nucleotide
fragment encoding the C-terminal 156 residues of PAPP-A2 with the stop codon replaced by a HindIII site for in-frame ligation into
the vector pcDNA3.1/Myc-His(-)A. The PCR product was
digested with EcoRI and HindIII and cloned into
the EcoRI/HindIII sites of the vector to generate
pPA2C-mH. The NotI-XbaI fragment and the
XbaI-EcoRI fragment were excised from pPA2 and cloned into
the NotI/EcoRI sites of pPA2C-mH. The resulting construct,
pPA2-mH, encoded PAPP-A2 followed by residues KLGP, the
c-myc epitope (EQKLISEEDL), residues NSAVD, and six
histidine residues.
pPA2-KO-mH--
A variant of pPA2-mH was constructed with a
Glu-734
Culture and transient transfection of human embryonic kidney
293T cells (293tsA1609neo) was performed as described earlier (6)
except that the cells were maintained for another 48 h in
serum-free medium (293 SFM II, Life Technologies, Inc.).
Measurement of PAPP-A2 Activity
A proteinase assay based on ligand blotting (15) with
radiolabeled IGF-II (Bachem) was used initially (for Fig. 3) to test for activity against IGFBP-1 (from HepG2-conditioned medium), rIGFBP-2
(GroPep), rIGFBP-3 (gift of D. Powell), rIGFBP-4 (Austral), rIGFBP-5
(gift of D. Andress), and rIGFBP-6 (Austral). For further analysis,
recombinant, c-myc- and His-tagged IGFBP-5 (rIGFBP-5) was
produced in mammalian cells. In brief, human placental oligo(dT)-primed cDNA (12) was used as a template to amplify cDNA encoding human IGFBP-5 (M65062). Primers containing an XhoI site
(5'-TCCGCTCGAGATGGTGTTGCTCACCGCGGT-3') and a HindIII site
(5'-CGATAAGCTTCTCAACGTTGCTGCTGTCG-3') were used, and the resulting PCR
product was cloned into the XhoI/HindIII sites of
pcDNA3.1/Myc-His(-)A (Invitrogen). Expression was
performed as above. Cleavage analysis was carried out by Western
blotting. Briefly, rIGFBP-5 as contained in cell culture medium (2 µl, ~10 ng of rIGFBP-5) was incubated with culture supernatants
from cells transfected with pPA2 or pPA2-KO (10 µl, ~2 ng of
rPAPP-A2) or with the same amount of culture supernatant from cells
transfected with empty vector. Phosphate-buffered solution and
inhibitors as specified in the main text were added to a final volume
of 25 µl. After incubation at 37 °C for 12 h, 10 µl of the
reaction mixture was separated by reducing 16% SDS-PAGE and blotted
onto a PVDF membrane, and intact rIGFBP-5 and the C-terminal cleavage product of rIGFBP-5 were detected with monoclonal
anti-c-myc (clone 9E10, ATTC) using
peroxidase-conjugated secondary antibodies (P260, DAKO), and ECL
(Amersham Pharmacia Biotech).
Miscellaneous Procedures
SDS-PAGE was performed in Tris/glycine gels (10-20% or 16%).
A metal chelate affinity column (1 ml, Amersham Pharmacia Biotech) was
charged with nickel ions and used for affinity purification of
His-tagged proteins in serum-free medium. Bound protein was eluted with
10 mM EDTA in phosphate-buffered solution containing 500 mM NaCl and further dialyzed against 20 mM
HEPES, 100 mM NaCl, and 1 mM CaCl2,
pH 7.4. For cleavage site determination, affinity-purified rIGFBP-5 (20 µg) was digested (37 °C for 12 h) with purified rPAPP-A2 (~1 µg) immobilized via anti-c-myc to recombinant
protein G-agarose (Life Technologies, Inc.). Edman degradation was
performed as described earlier (16). The amount of purified protein was
determined by amino acid analysis (17). Estimation of the amount of
rPAPP-A2 and rIGFBP-5 when non-purified was done by comparing the
responses in Western blotting (using anti-c-myc) with the
responses of known molar amounts of purified rIGFBP-5.
To ensure the complete absence of IGF-I or -II in the preparation of
rIGFBP-5, affinity-purified material (100 µg) was further loaded onto
a Superdex 75 (Amersham Pharmacia Biotech), equilibrated, and eluted
with 50% formic acid at 0.5 ml/min. At the acidity of the solvent
(pH < 1), any possible bound IGF would dissociate from rIGFBP-5
and elute separately (18, 19). Next, IGFBP-5-containing fractions were
loaded directly onto a reversed-phase high pressure liquid
chromatography (RP-HPLC) column (4 × 250 mm Nucleosil C4 500-7, Macherey-Nagel). A linear gradient was formed from 0.1% (v/v)
trifluoroacetic acid (solvent A) and 0.075% (v/v) trifluoroacetic acid
in 90% (v/v) acetonitrile (solvent B), increasing the amount of
solvent B by 2.3%/min. The column was equilibrated with 5% solvent B,
operated at 50 °C and with a flow rate of 0.5 ml/min, and the
separation was monitored at 226 nm. Bound IGFBP-5 eluted at ~42%
solvent B. The HPLC step alone was not sufficient as IGF-II (Bachem)
eluted close to IGFBP-5 on this column as seen in separate runs.
The pure protein was lyophilized, redissolved in solvent A, and further
diluted three times in 50 mM Tris, pH 7.5. This material
was prepared for experiments to demonstrate that cleavage of IGFBP-5 by
PAPP-A2 (using ~0.1 µg purified PAPP-A2/2 µg IGFBP-5) occurs in
the absence of IGF and is not promoted by IGF.
We have identified and cloned a novel cDNA sequence encoding a
protein of 1791 residues with homology to PAPP-A as detailed under
"Experimental Procedures". We name this protein
preproPAPP-A23. Alignment of preproPAPP-A2 with
preproPAPP-A (Fig. 1) demonstrates its
similarity to mature PAPP-A; there is no homology between the
prepro-peptides. In this alignment, 46% of the residues of mature
PAPP-A are also present in PAPP-A2. Like PAPP-A, the amino acid
sequence of PAPP-A2 contains three lin-notch motifs (LNR1-3) and five
short consensus repeats (SCR1-5) (Fig. 1). All 82 cysteine residues of
mature PAPP-A are also found in PAPP-A2, but PAPP-A2 has four
additional cysteines not present in PAPP-A. In addition, the zinc
binding site and a putative Met-turn are conserved between the two
proteins (Fig. 1), classifying PAPP-A and PAPP-A2 as metzincins (20).
Interestingly, the residue immediately following the third histidine of
the zinc binding consensus (Val-744 in PAPP-A2) and one residue close
to the methionine (Asn-805 in PAPP-A2) are conserved within each of the
four recognized metzincin superfamily members: the astacins, the
reprolysins, the serralysins, and the matrix metalloproteinases (20).
However, PAPP-A and PAPP-A2 do not fit in this pattern, they do not
show homology to any members of the four families, and further, the
linear distance between the third histidine and the methionine is much
longer in PAPP-A and PAPP-A2. PAPP-A and PAPP-A2 are therefore
reasonably classified as members of a new, fifth metzincin family,
which we tentatively designate the pappalysins.
To obtain recombinant PAPP-A2 (rPAPP-A2), 293T cells were transiently
transfected with an expression vector encoding c-myc tagged
PAPP-A2 (pPA2-mH). Western blotting of the culture medium showed a
single band of ~220 kDa (Fig. 2,
lane 2), absent in medium from mock transfected cells (Fig.
2, lane 1). Reduction of disulfide bonds did not cause a
visible change in band migration (Fig. 2, lane 4). Thus, in
contrast to PAPP-A, PAPP-A2 is not a disulfide bound dimer.
Pregnancy-associated Plasma Protein-A2 (PAPP-A2), a Novel
Insulin-like Growth Factor-binding Protein-5 Proteinase*
,,,,¶
Department of Molecular and Structural
Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C,
DK-8000 Aarhus C, Denmark and the § Endocrine Research Unit,
Mayo Clinic and Foundation, Rochester, Minnesota 55905
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Gln substitution; the NotI-KpnI
fragment of pPA2-KO was swapped into the
NotI-KpnI sites of pPA2-mH to generate
pPA2-KO-mH.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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View larger version (86K):
[in a new window]
Fig. 1.
Amino acid sequence of preproPAPP-A2
aligned with preproPAPP-A. The amino acid sequence of
preproPAPP-A23 (PA2) was aligned with the
sequence of preproPAPP-A2 (PA) using CLUSTALW
(29). Because the prepro-part of PAPP-A did not show significant
identity with the corresponding region of PAPP-A2, the alignment was
manually adjusted to emphasize difference in length of pro-peptides.
Arrows indicate the N-terminal residues of the mature
proteins as found earlier for PAPP-A (8) (Glu-81), and here for PAPP-A2
(Ser-234). Putative signal peptides, strongly predicted using SignalP
V2.0 (30), are shown with lowercase letters. The pro-part of
PAPP-A2 contains one other candidate initiation codon corresponding to
Met-168, but no signal peptide was predicted following this residue
using SignalP. The sequence motifs of PAPP-A (8) are also found in
PAPP-A2; the catalytic zinc binding motif (residues 733-743) and
residues of the putative Met-turn (around Met-807) are
underlined and bolded in both sequences.
Lin-notch motifs (LNR1-3) and short consensus repeats
(SCR-1-5) are boxed. All cysteines (shaded) of
mature PAPP-A are also found in PAPP-A2. In addition, the secreted form
of PAPP-A2 has four cysteine residues (Cys-343, Cys-533, Cys-618, and
Cys-1268) with no counterpart in PAPP-A.
View larger version (42K):
[in a new window]
Fig. 2.
Visualization of PAPP-A2 by Western blotting
and Coomassie staining. Medium from transfected 293T cells was
Western blotted using monoclonal anti-c-myc. Cells were
transfected with empty vector (lane 1), cDNA encoding
wild-type PAPP-A2 C-terminally tagged with the c-myc peptide
(pPA2-mH), (lane 2, non-reduced), or cDNA encoding
PAPP-A2 with an inactivating E734Q mutation (pPA2-KO-mH), (lane
3, non-reduced). Lane 4, as lane 2 but
reduced. Recombinant PAPP-A2 was purified by nickel affinity
chromatography from serum-free medium of cells transfected with
pPA2-KO-mH (lane 5, reduced, Coomassie-stained).
Edman degradation, performed on purified rPAPP-A2 (Fig. 2, lane 5) blotted onto a PVDF membrane, revealed the N-terminal sequence of the secreted rPAPP-A2 to be 234SPPEESNQ, corresponding to cleavage after the four residues RVKK233 (Fig. 1). This supports the prediction that PAPP-A2 is synthesized as a prepro-protein. The absence of an arginine residue in the P1 position indicates that the proprotein processing enzyme responsible for this cleavage is not furin but likely a different proprotein convertase (21).
Because the two motifs responsible for the metalloproteolytic activity
of PAPP-A are conserved in PAPP-A2, we hypothesized that PAPP-A2 is a
proteolytic enzyme, and because IGFBP-4 is the only known PAPP-A
substrate, we looked for a PAPP-A2 substrate among the six known IGFBPs
(Fig. 3). IGFBP-1, -2, -4, and -6 were not cleaved; IGFBP-3 showed limited degradation. However, complete proteolysis of IGFBP-5 is evident from this experiment.
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) or cDNA encoding PAPP-A2 (pPA2)
(+). The activity was assessed by ligand blotting using radiolabeled
IGF-II. Complete cleavage of IGFBP-5 is evident from the absence of a
signal in the BP5 (+) lane.
To further analyze the cleavage of IGFBP-5 by PAPP-A2, IGFBP-5 was
expressed in mammalian cells. Wild-type rPAPP-A2 (Fig. 4, lane 3), but not a variant
mutated in the zinc binding site (E734Q) (Fig. 4, lane 2),
efficiently degraded rIGFBP-5. As expected, 1,10-phenanthroline and
EDTA but not 3,4-dichloroisocoumarin abolished wild-type rPAPP-A2
activity (Fig. 4, lanes 4-6). Of interest, in contrast to
the proteolysis of IGFBP-4 by PAPP-A (1), cleavage of IGFBP-5 by
PAPP-A2 did not require the addition of IGF-I or -II. To verify that
cleavage was not induced by traces of IGF bound to IGFBP-5, we
further purified this by acidic gel filtration and (RP-HPLC). At low
pH, any bound IGF would dissociate from IGFBP-5 (18, 19) and elute
separately according to their different molecular weights. Using this
material, cleavage of IGFBP-5 by purified PAPP-A2 was still
observed without added IGF (Fig. 5, lane 2). Interestingly, added IGF-II did not have any
apparent effect on the rate of cleavage as seen with shorter incubation resulting in incomplete digestion (Fig. 5, lanes 3 and
4).
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For cleavage site determination, purified rIGFBP-5 (Fig.
6, lane 1) was digested with
purified PAPP-A2 and analyzed by SDS-PAGE (Fig. 6, lane 2).
Edman degradation of blotted material showed that both
distinct, visible degradation products (Fig. 6, lane 2)
contained the N-terminal sequence
144KFVGGA4. Both
of the two distinct bands represent intact C-terminal cleavage fragments because they also contain the C-terminal c-myc tag
(Fig. 6, lane 3); they are likely to be differently
glycosylated in accordance with the heterogeneity of purified rIGFBP-5
(Fig. 6, lane 1). Both bands contained a second sequence at
lower level (45%), 1LGXFVH, corresponding to the
N-terminal sequence of IGFBP-5. The absence of Ser, expected in the
third cycle of Edman degradation, was taken as evidence for
carbohydrate substitution of Ser-3. O-linked glycan on the
N-terminal cleavage fragment is likely to cause it to smear around the
two distinct, C-terminal fragments. Sequence analysis directly on the
reaction mixture (>100 pmol) without SDS-PAGE separation showed only
the same two IGFBP-5 sequences in equimolar amounts. Thus, PAPP-A2
cleaves IGFBP-5 at one site between Ser-143 and Lys-144.
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Probing a blot with mRNA from several human tissues, previously used for PAPP-A (12), resulted in a signal from the human placenta only (not shown). However, using the blast algorithm (13), a total of 98 human EST sequences were identified that matched the 3' untranslated region of the PAPP-A2 mRNA sequence. Of these, 39% originated from placenta, 21% from pregnant uterus, 11% from fetal liver/spleen, and 5% from kidney. Several other tissues were represented but with fewer EST sequences. Hence, like PAPP-A (12), PAPP-A2 expression is neither limited to the placenta nor to pregnancy.
Proteolytic activity against IGFBP-5 has been widely reported from several sources, e.g. pregnancy serum (22), seminal plasma (23), culture media from smooth muscle cells (24), granulosa cells (25), osteosarcoma cells (26), and also from osteoblasts (27) and fibroblasts (28). Matrix metalloproteinase-2 and the serine proteinase complement C1s have been reported to contribute to the IGFBP-5 proteolytic activity in media from osteoblasts (27) and fibroblasts (28), respectively. However, in general, the proteinase responsible for cleavage of IGFBP-5 has remained unidentified.
The recent identification of PAPP-A as the IGFBP-4 proteinase in fibroblasts (1), osteoblasts (1, 4), ovarian follicular fluid (7), pregnancy serum (6), and vascular smooth muscle cells (5) firmly establishes PAPP-A and IGFBP-4 as an important functional pair in several systems. No other substrate has been found for PAPP-A, and no other proteinase has been shown to cleave IGFBP-4 physiologically. It is therefore very tempting to speculate that the pair of PAPP-A2 and IGFBP-5 plays an analogous role in a number of the tissues mentioned above. Interestingly, incubating IGFBP-5 with smooth muscle cells-conditioned medium resulted in cleavage between Ser-143 and Lys-144 (24), the same cleavage site as found here with purified PAPP-A2. This immediately identifies PAPP-A2 as an obvious candidate IGFBP-5 proteinase for this tissue.
After completion of the experimental work presented here, data base searching revealed that additional genomic sequences (AC027620 and AL139282) and a partial cDNA sequence (AJ278348) had appeared. From none of these, however, can the complete cDNA sequence of PAPP-A2 be deduced.
In conclusion, we have identified, cloned, and expressed a novel
protein with homology to PAPP-A, and we have demonstrated the putative
proteolytic activity of this protein. We named the protein PAPP-A2 to
signify its close relationship with PAPP-A both structurally and
functionally. With PAPP-A, PAPP-A2 defines a new, fifth family of the
metzincin superfamily of metalloproteinases, the pappalysins. Further,
we have identified a natural substrate for PAPP-A2, IGFBP-5, analyzed
its cleavage, and indicated several tissues where PAPP-A2 may be of
physiological relevance.
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Note Added in Proof |
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Following the acceptance of this manuscript we have become aware that the partial sequence of database entry AJ278348 has been presented in a publication (Farr et al., (2000) Biochim. Biophys. Acta 1493, 356-362). In this paper, residues 147-1791 of PAPP-A2 is named PAPP-E.
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FOOTNOTES |
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* This work was supported by grants from the Danish Medical Research Council, the Novo Nordic Foundation, and the Alfred Benzon Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF311940.
¶ To whom correspondence should be addressed: Fax: 45-8612-3178; E-mail: co@mbio.aau.dk.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M102191200
3 The entire sequence of 5376 nucleotides encoding preproPAPP-A2 (1791 residues) is deposited in the GenBankTM data base under accession number AF311940.
2 The numbering of preproPAPP-A (GenBankTM accession number AAC50543) (9) is used in this paper.
4 IGFBP-5 is numbered with the N-terminal Leu of the mature protein as residue 1.
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ABBREVIATIONS |
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The abbreviations used are: PAPP-A, pregnancy-associated plasma protein-A; PAPP-A2, pregnancy-associated plasma protein-A2; IGFBP, insulin-like growth factor-binding protein; IGF, insulin-like growth factor; proMBP, the proform of eosinophil major basic protein; PCR, polymerase chain reaction; nt(s), nucleotide(s); EST, expressed sequence tag; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; RP-HPLC, reversed-phase high pressure liquid chromatography.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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