Molecular Human Reproduction vol.5 no.10 pp. 983–989, 1999
Alternative forms of a novel aspartyl protease gene are
differentially expressed in human gestational tissues
E.K.Moses1, K.A.Freed, J.R.Higgins and S.P.Brennecke
Department of Perinatal Medicine, The Royal Women’s Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia
1To
whom correspondence should be addressed
The aim of this study was to identify genes involved in human placentation. To do this, differential gene
expression was assessed in the decidua (placental bed) from pre-eclamptic and normotensive pregnancies
using the polymerase chain reaction (PCR)-based subtractive technique of representational difference analysis.
A novel aspartyl protease (cathepsin D-like) cDNA sequence was isolated by virtue of its over-expression in
the pre-eclamptic decidual sample tested. It was designated DAP-1 (for Decidual Aspartyl Protease 1). Using
DAP-1 primer sequences a second cDNA (DAP-2) was subsequently isolated from decidual RNA by reverse
transcription (RT)–PCR and found to be identical to DAP-1 apart from 80 additional and consecutive base
pairs in the N-terminal coding region. In DAP-2, a stop codon within the unique 80 bp sequence was predicted
to terminate translation immediately before the consensus active site residues. While Southern blotting was
used to show that there are two loci with homology to DAP-1 in the human genome, it is postulated that
alternative pre-mRNA splicing of the 80 bp exon is involved in the regulated expression of active (DAP-1) and
inactive (DAP-2) forms of this novel protease; a mechanism similar to that involved in the regulated expression
of Caspase-2, a protease involved in apoptosis. In other systems the regulation of alternative splicing is
indicated by tissue specificity and developmental stage specificity of the various spliced products. In this
context it was demonstrated that whereas DAP-1 was the major transcript expressed in decidua, the pattern
was reversed in the adjacent placental tissue. It is proposed that tissue and developmental stage-specific
expression of the DAP protease are important for the normal development and function of the uteroplacental
tissues and that dysregulation of the control of DAP gene splicing may play a role in abnormal placentation,
like that seen in pre-eclampsia.
Key words: mRNA expression/placentation/pregnancy/representational difference analysis
Introduction
Human placentation involves a complex interaction between
fetal and maternal tissues. While several proteins that play a
role in early placentation have been identified, a complete
understanding of the cellular and molecular components
involved is just beginning (Cross et al., 1994; Kliman, 1994).
A central feature of human placentation involves the formation
of columns of cytotrophoblasts that invade the maternal uterine
tissues and in particular the maternal decidual vasculature.
This invasion extends along the spiral arteries, through the
full thickness of the decidua, to the inner third of the
myometrium. The cytotrophoblasts replace the endothelial cell
lining of the maternal spiral arteries and destroy the underlying
extracellular matrix and vascular smooth muscle. These spiral
arteries are thus transformed into large diameter vessels of
low resistance which maximizes maternal blood flow into the
intervillous space (Cross et al., 1994; Kliman 1994; Zhou
et al., 1997a).
Abnormal placentation with shallow trophoblastic spiral
artery invasion and inadequate maternal spiral artery transformation is a characteristic feature of pre-eclampsia, the
most common serious medical disorder of human pregnancy
(Robertson et al., 1986; Zhou et al., 1997b). It is postulated
© European Society of Human Reproduction and Embryology
that, in pre-eclampsia, failure of spiral artery transformation
subsequently leads to placental hypoxia and this in turn leads
to the release by the placenta of a factor(s) which mediates
widespread maternal endothelial cell dysfunction (Higgins and
Brennecke, 1998). In an attempt to elucidate molecular events
associated with abnormal placentation we used a new molecular
technique, called representational difference analysis (cDNA–
RDA; Hubank and Schatz, 1994), to examine differential gene
expression in normal and pre-eclamptic decidual (placental
bed) tissues. In this paper we report the characterization of
novel aspartyl protease gene sequences that were identified
using this approach.
Materials and methods
Reagents
Acrylamide, herring sperm DNA and morpholinopropane-sulphinic
acid (MOPS) were purchased from Roche Diagnostics, Australia.
Agarose, bisacrylamide, deoxynucleotide triphosphates and pGEM
DNA markers were obtained from Promega Corporation, Castle Hill,
NSW, Australia. Guanidine thiocyanate was purchased from Fluka
(Buchs, Switzerland). Phenol (Wako special grade) was obtained from
Novachem (Melbourne, Australia). The oligonucleotides described in
the original protocol by Hubank and Schatz (1994) were synthesized
983
E.K.Moses et al.
at Monash University, Melbourne, Australia. Mung Bean Nuclease
and T4 DNA ligase were bought from Promega, Australia. DpnII was
obtained from New England Biolabs. All polymerase chain reactions
(PCR) reactions were performed using 5 U of Taq DNA polymerase
(Gibco BRL) in 13PCR buffer (Gibco BRL; 10 mmol/l Tris–HCl
pH 8.3, 50 mmol/l KCl, 4 mmol/l MgCl2). Glycogen and yeast RNA
was purchased from Boehringer Mannheim. DNA extraction from
agarose gels was carried out using a Quiex gel extraction kit (Qiagen),
according to the manufacturer’s instructions.
Sample collection
Placental bed biopsies (decidua) were obtained (with Institutional
Research and Ethics Committee approvals) during Caesarean
section from eleven normal patients and one patient with preeclampsia. The tissues were immediately frozen in liquid nitrogen
and stored at –80°C until processed. The patient with pre-eclampsia
was diagnosed according to internationally agreed criteria (Davey
and MacGillivray, 1988). Eight placentae were collected from women
who delivered singleton infants by elective Caesarean section at term.
Within 10 min of delivery, placental tissue was collected and RNA
was immediately extracted.
RNA isolation and preparation of cDNA
Total cytoplasmic RNA was isolated by a guanidinium thiocyanate/
phenol chloroform method (Chomczynski and Sacchi, 1987). The
quantity of RNA recovered was established spectrophotometrically
and its integrity assessed by electrophoresis on a 1% non-denaturing
agarose gel (Sambrook et al., 1989). PolyA1 mRNA was isolated
from total RNA using a commercial system (PolyATtract mRNA
isolation kit, Promega). Double-stranded cDNA was prepared by
reverse transcription (RT) of the polyA1 mRNA using the RiboClone
cDNA Synthesis Systems AMV RT kit (Promega), according to the
manufacturer’s instructions.
RDA of cDNA
RDA of cDNA involves the digestion of similar cDNA populations
with the restriction enzyme DpnII followed by adaptor ligation
and PCR-mediated amplification of the restriction fragments. If
an amplifiable restriction fragment (the target gene) exists in one
representation of cDNA (the Tester) and is either absent or in lower
abundance in another (the Driver), a kinetic enrichment of the target
is achieved by subtractive hybridization of the Tester in the presence
of excess Driver. Successive iterations of the subtractive hybridization/
PCR amplification produce ethidium visible bands on an agarose gel
corresponding to the enriched target. Difference products can be
excised from the gel, and cloned into a plasmid vector for detailed
molecular characterization. To selectively amplify genes that are upregulated in pre-eclampsia, a pool of cDNA prepared from three
normal placental bed tissues was used as the Driver; the Tester was
cDNA prepared from placental bed tissue from a patient with preeclampsia. RDA of cDNA was implemented according to the method
of Hubank and Schatz (1994), using the Tester and Driver cDNA
described above. Briefly, three rounds of hybridization/amplification
were carried out, generating difference products 1, 2 and 3 (DP1,
DP2 and DP3). The ratios of Driver to Tester for DP1, 2 and 3
were 100:1, 800:1 and 400 000:1 respectively. All procedures were
performed using plugged filter tips (ART; Continental Laboratory
Products, San Diego, USA) and the PCR-mediated amplification was
carried out in an OMNE thermocycler (Hybaid) using 0.5 ml tubes.
Cloning Kit, Stratagene, La Jolla, CA, USA], according to the
manufacturer’s instructions. Double-stranded plasmid DNA were
prepared from transformed E.coli cultures by the alkaline–sodium
dodecyl sulphate (SDS) lysis procedure (Sambrook et al., 1989).
Sequencing reactions were performed on recombinant plasmids using
a Dye Terminator kit (Perkin–Elmer, Norwalk, CT, USA). Sequencing
analysis was performed using an Applied Biosystems ABI 377
automated sequencer (Perkin–Elmer, Norwalk, CT, USA). Sequence
comparisons were made with sequences in GenBank using the
computer program BLAST, residing at the Australian National Genomic Information Service (http://www.angis.org.au).
Northern blot analysis
Total RNA (20 µg) was denatured in formamide (35% v/v) and
formaldehyde (12% v/v) for 10 min, size-fractionated on a 1%
denaturing agarose gel and then capillary-blotted onto Hybond N
membrane (Amersham, Castle Hill, NSW, Australia), as described
previously (Freed et al., 1995). An RNA ladder spanning 0.24–9.5
kb (Gibco BRL, Gaithersburg, MD, USA) was run on the gel to
estimate transcript size. The cloned 253 bp cDNA fragment (25 ng)
derived from DP3 (DAP-1) was radiolabelled with [α-32P]dCTP
(Amersham: 3000 Ci/mmol) using a random primer oligolabelling
kit (Prime-a Gene® Labelling Kit; Promega Corporation). Membranes
were prehybridized at 42°C for 3 h in hybridization buffer containing
50% formamide, 53SSPE (0.7 mol/l NaCl, 0.04 mol/l NaH2PO4·H2O,
0.005 mol/l EDTA pH 7.4), 53Denhardt’s solution [13Denhardt’s
contains 0.02% (w/v) each of BSA, Ficoll 400 and polyvinylpyrrolidone], 0.5% SDS and 0.5 mg/ml denatured herring sperm DNA.
Hybridization of the radiolabelled probe to the membrane was carried
out for 16 h at 42°C in hybridization buffer. The membrane was
washed initially with 23SSC (SSC consists of 0.15 mol/l NaCl,
0.017 mol/l sodium citrate pH 7.0)/ 0.1% SDS at room temperature
(15 min), followed by a wash in 0.13SSC/0.1% SDS at 50°C.
Autoradiographs were obtained by exposure of the membrane to
Kodak X-Omat AR film at –80°C.
Multiple tissue Northern blot
A commercial human multiple tissue Northern blot (Clontech) was
probed with the 250 bp radiolabelled DAP-1 cDNA (described above)
using the buffers and hybridization conditions provided by the
manufacturer.
Southern blotting
Zooblot
Human, sheep, rabbit, rat, mouse, chicken, lizard and frog DNA was
a generous gift from Dr J.Hendry (Monash Medical Centre, Victoria,
Australia). DNA (10 µg) was digested with EcoRI for 6 h at 37°C.
In addition, human DNA (10 µg) was digested separately with
HindIII, PstI and BamHI, for 6 h at 37°C. The DNA fragments were
then loaded onto a 1% agarose gel and electrophoresed overnight in
13TAE buffer. The size-fractionated DNA was Southern-blotted to
Hybond N membrane (Amersham). The membrane was baked for
2 h at 80°C then prehybridized at 42°C in hybridization buffer.
DAP-1 cDNA was radiolabelled as for the Northern blot analysis.
Hybridization was performed overnight at 42°C in the same solution
as for prehybridization with the exception that the 53Denhardt’s
solution was omitted. The membrane was washed initially in 23SSC/
0.1% SDS at room temperature, then increasing stringency up to
0.53SSC/0.1% SDS at 65°C.
RT–PCR
Cloning and sequencing
cDNA fragments from DP3 were cloned blunt-ended into the Srf
I site of the pCR-ScriptTMSK(1) phagemid [pCR-Script™SK(1)
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Primer design
The synthetic oligonucleotide primers that were used for semiquantitative RT–PCR analysis of DAP-1 were: forward, 59-GC-
Novel aspartyl protease genes and placentation
AGGGCACACTGAAGAAGT-39; and reverse, 59-GGATGGAGAGAACCAGCAGA-39. These primers anneal to the corresponding
(100% identical) region of DAP-2.
Reverse transcription
Total RNA samples used in RT–PCR experiments were treated with
Proteinase K (100 ng/µl; Boehringer Mannheim) and then with RQ1
RNase-free DNase (0.2 U/µl; Promega) to remove contaminating
chromosomal DNA. RNA (400 ng) was reverse-transcribed in a
20 µl reaction containing 200 U MMLV RT (Gibco BRL),
10 µmol/l oligo (dT)15 primer (Promega Corporation), 500 µmol/l
each of dATP, dCTP, dGTP and dTTP, 10 U RNAase inhibitor
(Promega), 10 mmol/l DTT and 13reverse transcriptase buffer (Gibco
BRL; 50 mmol/l Tris–HCl pH 8.3, 75 mmol/l KCl, 3 mmol/l MgCl2),
at 35°C for 1 h. The reaction was terminated by incubation at 95°C
for 5 min.
PCR
PCR-mediated amplification was carried out in a 20 µl reaction
containing 1 µl from each RT reaction, 1 µmol/l each of the forward
and reverse primers, 200 µmol/l each of dATP, dCTP, dGTP and
dTTP, 2.5 U Taq DNA polymerase (Gibco BRL), 13PCR buffer
(Gibco BRL; 10 mmol/l Tris–HCl pH 8.3, 50 mmol/l KCl) and either
1.5 (for decidual samples) or 1.8 mmol/l MgCl2 (placental samples).
The reactions were overlaid with mineral oil (50 µl) and for decidual
samples PCR was performed for 25 cycles (95°C, 1 min; 60°C,
1 min; 72°C, 1 min) using an ITC 100 Immersion Thermocycler
(Bartelt Instruments, Australia). Placental samples were amplified
using the same thermocycling profile for 35 cycles.
Southern blotting
Samples of each RT–PCR reaction (20% v/v ) were size-fractionated
by electrophoresis on a 1.5% agarose gel in TAE buffer (0.04 mol/l
Tris acetate, 0.001 mol/l EDTA, pH 8.4) followed by capillary
blotting to Hybond N nylon membrane (Amersham) (Sambrook
et al., 1989). DNA size markers (pGEM DNA markers, Promega
Corporation) were run in parallel to estimate fragment size.
Membranes were prehybridized at 42°C for 3 h in the hybridization
buffer described previously. 32P-radiolabelled DAP-1 cDNA (as used
for Northern analysis) was used as a probe to detect both DAP-1 and
DAP-2 RT–PCR products.
Results
Cloning of novel aspartyl protease cDNA
For our RDA study we isolated RNA from decidual biopsies
taken from normal and pre-eclamptic placental beds. The RNA
from three normal tissues were pooled and used to prepare a
Driver cDNA representation. The Tester cDNA representation
was derived from a single pre-eclamptic tissue. Difference
products (DP1, DP2 and DP3) were obtained at the end of
each of the three iterative cycles (Figure 1).
To facilitate molecular characterization, the major 347 bp
cDNA amplicon in DP3 was purified from agarose gel and
cloned into the plasmid vector pCRScript. The nucleotide
sequence of the cloned cDNA fragment was determined and
found to have no similarity with any sequences in the databases.
In Northern blot analysis, using this cloned cDNA as a
radiolabelled probe, no transcripts could be detected in any
of the four tissue RNA used for RDA of cDNA (results
not shown).
Figure 1. Representational difference analysis (RDA) of cDNA.
Driver (D) and Tester (T) cDNA representations were prepared
from normal (n 5 3) and pre-eclamptic (n 5 1) decidual RNA
samples respectively. Difference products (DP1, DP2 and DP3)
were obtained after each of the three iterative cycles. Samples from
each step were subjected to agarose gel electrophoresis.
A second 253 bp cDNA amplicon was cloned from DP3 and
found to predict a polypeptide sequence with 95% similarity to
the N-terminal region of an aspartyl protease recently cloned
from mouse kidney (kap, Mori et al., 1997) and 76% similarity
with the corresponding region of human cathepsin D (Faust
et al., 1985). This N-terminal region contains the consensus
catalytic site residues DTGSSNLW of aspartyl proteases
(Figure 2). Accordingly, the 253 bp cDNA amplicon was
designated DAP-1 (for Decidual Aspartyl Protease 1).
In RT–PCR reactions (using internal DAP-1 primers) subsequently performed on decidual RNA (including those used for
RDA), we found that a 289 bp cDNA amplicon (additional to
the expected 209 bp DAP-1 fragment) was just visible in some
reactions (shown later in Figure 5). Cloning and sequence
analysis of this larger cDNA amplicon (DAP-2) revealed that
it was identical to the DAP-1 cDNA amplicon, apart from an
additional 80 bp sequence in the 59 end. This 80 bp sequence
contains a stop codon that is in-frame with the putative protease
coding sequence whereby the predicted polypeptide is truncated
just short of the conserved catalytic site residues (Figure 3).
To verify the putative up-regulation of DAP-1, Northern
blot analysis was performed on the decidual RNA samples
used for RDA of cDNA. A 1.5 kb transcript was found to be
abundantly expressed in the Tester RNA sample but only
weakly expressed in one of the three Driver RNA (Figure 4).
This transcript is similar in size to the mouse kap mRNA
(1.6 kb; Mori et al., 1997) whereas the human cathepsin D
mRNA is ~2.2 kb (Faust et al., 1985). It was considered
appropriate to use DAP-1 as a probe since DAP-2 was only
just detectable (in some decidual samples, including those
used for RDA) by the far more sensitive RT–PCR methodology
(shown later in Figure 5).
Differential expression of DAP-1 and DAP-2 in human
uteroplacental tissues
Using primer sequences common to both DAP-1 and DAP-2
the relative expression of these transcripts in normal placental
985
E.K.Moses et al.
Figure 2. Sequence alignment of the putative DAP-1 polypeptide sequence with the corresponding N-terminal sequences of the recently
identified novel mouse aspartyl protease kap, human cathepsin D and human renin.
Figure 3. Sequence analysis of DAP-1 and DAP-2. (A) The nucleotide sequence of DAP-1. The putative DAP-1 polypeptide sequence is
shown above. (B) The additional 80 bp sequence from DAP-2; its point of insertion in DAP-1 indicated by n. The alternative polypeptide
extension, predicted from insertion of the unique 80 bp DAP-2 sequence, terminating (*) at a TGA stop codon.
(n 5 8) and decidual (n 5 12) tissues was determined
by semi-quantitative RT–PCR. In decidua, DAP-1 is the
predominant transcript whereas in the placenta the pattern is
reversed (Figure 5).
Expression of DAP in other human tissues
Hybridization of the radiolabelled DAP-1 cDNA probe to a
commercial human multiple tissue Northern blot (Clontech)
revealed that a 1.5 kb transcript was expressed most abundantly
in the lung and moderately in the kidney. With the possibility
of very weak expression in the heart no expression was
detected in the other tissues examined (pancreas, smooth
muscle, liver, placenta, and brain; Figure 6). This restricted
expression profile for DAP is similar to that reported for kap
in the mouse (Mori et al., 1997) and is in contrast with the
ubiquitous distribution of cathepsin D mRNA expression (Faust
et al., 1985; Tang and Wong, 1987).
Figure 4. Northern blotting of the normal (N1, N2, N3) and preeclamptic (PE) decidual RNA samples used to prepare the Driver
and Tester cDNA representations for RDA. The blot was
hybridized with radiolabelled DAP-1 cDNA. The position and size
of standard RNA markers that were run in parallel are indicated.
986
DAP sequences are conserved across species
Southern blot analysis revealed that DAP sequences are highly
conserved in nature. Hybridization was detected in the genomes
of the sheep, rabbit, rat, mouse, chicken lizard and frog (Figure
7A). Southern blots of human genomic DNA that had been
digested independently with several different restriction
endonucleases radiolabelled with a DAP-1 probe (Figure 7B).
Since the probe contained no restriction sites for these endonucleases, the detection of two bands supports the notion that
the cloned DAP-1 and DAP-2 cDNA are coded for by
separate genes.
Novel aspartyl protease genes and placentation
Figure 5. Semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of DAP-1 and DAP-2 mRNA expression in
normal placentae (n 5 8) and decidua (n 5 12). RT–PCR was performed using the internal DAP-1 primers also common to DAP-2.
Following agarose gel electrophoresis and Southern blotting, the membrane was hybridized with radiolabelled DAP-1 cDNA. The two
normal (N1, N2) and one pre-eclamptic (PE) samples used in the cDNA representational difference analysis are indicated.
Figure 6. DAP mRNA expression in human tissues. A commercial
human multiple tissue Northern blot (Clontech) was probed with
radiolabelled DAP-1 cDNA. The position and size of standard RNA
markers that were run in parallel are indicated.
Discussion
The complex molecular processes that contribute to the
physiology and pathology of human placentation are poorly
understood. Differential gene expression is likely to play a
key role but, until recently, the techniques available to study
differential gene expression have required relatively large
quantities of RNA and have been of questionable utility for
the isolation of minor differences. RDA offers a new approach
to the assessment of differential gene expression (Lisitsyn
et al., 1993). Originally developed for the identification of
differences between complex genomic DNA populations, it
has since been adapted for the analysis of differential gene
expression (cDNA-RDA; Hubank and Schatz, 1994). This
procedure is sensitive, results in the isolation of very few
false-positive clones and opens the possibility of finding novel
expressed genes.
The majority of studies utilizing cDNA–RDA have investigated differential gene expression in cell lines (Niwa et al.,
1997; Arvand et al., 1998; Chang et al., 1998; Chu and Paul,
1998; Lawson and Berliner, 1998; Lawson et al., 1998).
However, relatively fewer studies have adopted this technique
to study differential gene expression in tissues (Lerner et al.,
1996; Gruidl et al., 1997; Wada et al., 1997; Welford et al.,
1998). We have now successfully applied cDNA–RDA to
isolate a novel aspartyl protease gene sequence DAP-1, by
virtue of the over-expression of this gene in the decidua from
a severe case of pre-eclampsia. This is the first reported use
of RDA in obstetric research and the first molecular cloning
of an aspartyl protease cDNA from uteroplacental tissues. We
suggest that this technique could be used in a variety of
reproductive studies to identify candidate genes.
The aspartyl proteases are a family of proteolytic enzymes
that share a unique catalytic apparatus and mechanism, having
apparently evolved from the same ancestral gene (Tang, 1979;
Tang and Wong, 1987). These enzymes usually function in
acidic environments, a characteristic that limits their function
to some specific locations in different organisms. Thus, the
best known sources of mammalian aspartic proteases are
stomach (for pepsin, gastricsin and chymosin), kidney (for
renin), lysosomes (for cathepsin D) and endoplasmic reticulum
(for cathepsin E). The function of these enzymes includes the
digestion of proteins such as casein and clot milk (for gastric
aspartic proteases), the biosynthesis of angiotensin I from
angiotensinogen (for renin), and the degradation of intracellular
and endocytosed proteins (for cathepsin D). The physiological
function of cathepsin E is currently unknown.
The physiological function(s) of the putative DAP-1 protease
is not known and is the subject of our continued investigation.
Cathepsin D, the most similar (of the human aspartyl proteases)
has multiple functions in different cellular compartments,
including endosomal proteolysis of endocytosed proteins (Tang,
1979; Tang and Wong, 1987), proteolytic activation (also
endosomal) of secreted proteins such as growth factors and
hormones (Diment et al., 1989) and cytokine-induced apoptosis
(Deiss et al., 1996). In addition a secreted form of cathepsin
D has been implicated in controlling the migration of metastatic
cells (Mignatti and Rifkin, 1993) and could also be involved
in the control of cell migration during normal development
(Deiss et al., 1996). The restricted distribution of DAP mRNA
expression to primarily lung and kidney (as shown by Northern
blot analysis) is not in keeping with the ubiquitous distribution
of cathepsin D mRNA expression (Tang and Wong 1987).
Rather, the distribution of DAP mRNA expression is in good
agreement with the restricted distribution (primarily kidney
and lung) of the recently cloned (and most similar) mouse kap
mRNA, whose function is unknown (Mori et al., 1997).
Cathepsin D is expressed in the (non-pregnant) endometrium
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E.K.Moses et al.
Figure 7. Southern blot analysis. (A) Human genomic DNA digested separately with various restriction endonucleases (HindIII, PstI,
BamHI, EcoRI) hybridized with radiolabelled DAP-1 cDNA. There were no sites for enzymes within the DAP-1 cDNA probe. (B) Zooblot
of EcoRI digested genomic DNA hybridized with radiolabelled DAP-1 cDNA.
where its levels are increased in response to progesterone
(Maudelonde et al., 1990; Bergqvist et al., 1996; Camier et al.,
1996) but, as far as we are aware, there are no reports of a
role for this protease in placentation.
With respect to the involvement of aspartyl proteases during
pregnancy, renin is the only enzyme to have been the subject
of detailed investigation in human gestational tissues. The
potential for a locally acting renin–angiotensin system (RAS)
to moderate regional haemodynamics and other organ functions
by virtue of autocrine and/or paracrine actions has led to
speculation that the impaired uteroplacental blood flow in preeclampsia may be related to alterations of the placental (locally
acting) RAS. In this context it was found that there was a
tendency toward increased expression of the renin gene in the
placental extracts from pre-eclamptic pregnancies (Sowers
et al., 1993). However, although the existence of a complete
RAS in the pregnant human uterus is generally accepted, some
investigators have recently suggested that this long-held belief
needs to be clarified, due to the apparent lack of (or unknown)
specificity of the antibody and nucleic acid probes used to
detect renin in uteroplacental tissues. In particular, the crossreactivity of renin antibody probes with cathepsin D has raised
988
the possibility that this, or another aspartyl protease(s), may
be present in these tissues (Hanssens et al., 1995a,b). Since
the putative DAP protease has extensive sequence similarity
to both renin and cathepsin D (72 and 76%, respectively) it is
possible that all three enzymes could be candidates for the
molecules previously detected in gestational tissues with renin
probes. This remains to be elucidated.
The DAP-1 and DAP-2 sequences isolated in this study are
either coded for by separate genes or are derived from the
same gene via alternative splicing. While our Southern data
(Figure 7) show that there are two homologous DAP gene loci
in the human genome, there are features in common with other
alternatively spliced genes that lead us to speculate on the
involvement of this latter mechanism in the tissue-specific
expression of active and inactive forms of the DAP protease.
In particular, the premature termination of translation predicted
by the DAP-2 sequence (caused by the 80 bp sequence
additional to that seen in DAP-1) has striking similarity with
the reported mechanism for regulation of the caspase-2 gene
(ICH-2). For ICH-2, the alternative inclusion or exclusion of
a 61 bp exon is involved in the production of two transcripts,
ICH-1S and ICH-1L respectively. Inclusion of the 61 bp exon
Novel aspartyl protease genes and placentation
results in translation termination using a more upstream stop
codon in Ich-1S generating a shorter inactive protein product
(Jiang and Wu, 1999).
Although some alternative splicing events appear to be
constitutive, with mRNA variants coexisting at constant ratios
in the same cells, this mechanism is also widely involved in
the regulation of tissue and developmental stage-specific gene
expression (Lopez, 1998; Jiang and Wu, 1999). Not surprisingly, some human genetic diseases and developmental defects
have been correlated with disruptions of alternative splicing
control in particular genes (Lopez, 1998). It is possible that
tissue and developmental stage-specific expression of the DAP
protease are important for the normal development and function
of the uteroplacental tissues and that dysregulation of the
control of DAP gene splicing may play a role in abnormal
placentation, like that seen in pre-eclampsia.
Acknowledgements
The authors gratefully acknowledge the assistance of clinical research
midwives Linda Horton and Nicola Davies and the obstetric and
midwifery staff of the Royal Women’s Hospital, Melbourne, Australia,
for their cooperation. This study was funded by a grant from the
3AW Community Services Trust Fund of the Royal Women’s Hospital,
Melbourne, Australia.
References
Arvand, A., Bastians, H., Welford, S.M. et al. (1998) EWS/FLI1 regulates
ME2-C, a cyclin-selective ubiquitin conjugating enzyme involved in cyclin
B destruction. Oncogene, 17, 2039–2045.
Bergqvist, A., Ferno, M. and Mattson, S. (1996) A comparison of cathepsin
D levels in endometriotic tissue and in uterine endometrium. Fertil. Steril.,
65, 1130–1134.
Camier, B., Hedon, B., Rochefort, H. et al. (1996) Endometrial cathepsin D
immunostaining throughout ovulatory and anovulatory menstrual cycles.
Hum. Reprod., 11, 392–397.
Chang, D.D., Park, N.H., Denny, C.T. et al. (1998) Characterization of
transformation related genes in oral cancer cells. Oncogene, 16, 1921–1930.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation
by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal.
Biochem., 162, 156–159.
Cross, J.C., Werb, Z., and Fisher, S.J. (1994) Implantation and the placenta:
key pieces of the development puzzle. Science, 266, 1508–1518.
Chu, C.C. and Paul, W.E. (1998) Expressed genes in interleukin-4 treated B
cells identified by representational difference analysis. Mol. Immunol., 35,
487–502.
Davey, D.A. and MacGillivray. I. (1988) The classification and definition of
the hypertensive disorders of pregnancy. Am. J. Obstet. Gynecol., 158,
892–898.
Deiss, L.P., Galinka, H., Berissi, H. et al. (1996) Cathepsin D protease
mediates programmed cell death induced by interferon-gamma, Fas/APO1 and TNF-alpha. EMBO J., 15, 3861–3870.
Diment, S., Martin, K.J. and Stahl, P.D. (1989) Cleavage of parathyroid
hormone in macrophage endosomes illustrates a novel pathway for
intracellular processing of proteins. J. Biol. Chem., 264, 13403–13406.
Faust, P.L., Kornfeld, S. and Chirgwin, J.M. (1985) Cloning and sequence
analysis of cDNA for human cathepsin D. Proc. Natl. Acad. Sci. USA, 82,
4910–4914.
Freed, K.A., Aitken, M.A., Brennecke, S.P. et al. (1995) Prostaglandin G/
H synthase-1 messenger RNA relative abundance in human amnion,
choriodecidua and placenta before, during and after spontaneous-onset
labour at term. Gynecol. Obstet. Invest., 39, 73–78.
Gruidl, M., Buyuksal, A., Babaknia, A. et al. (1997) The progressive rise in
the expression of alpha crystallin B chain in human endometrium is initiated
during the implantation window: modulation of gene expression by steroid
hormones. Mol. Hum. Reprod., 3, 333–342.
Hanssens, M., Vercruysse, L., Verbist, L. et al. (1995a) Renin-like
immunoreactivity in human placenta and fetal membranes. Histochem. Cell.
Biol., 104, 435–442.
Hanssens, M., Vercruysse, L., Keirse, M.J. et al. (1995b) Identification of
‘renin’-containing cells in the choriodecidua. Placenta, 16, 517–525.
Higgins, J.R. and Brennecke, S.P. (1998) Pre-eclampsia — still a disease of
theories? Curr. Opin. Obstet. Gynaecol., 10, 129–133.
Hubank, M. and Schatz, D.G. (1994) Identifying differences in mRNA
expression by representational difference analysis of cDNA. Nucl. Acids
Res., 22, 5640–5648.
Jiang, Z-H. and Wu, J.Y. (1999) Alternative splicing and programmed cell
death. Proc. Soc. Exp. Biol. Med., 220, 64–72.
Kliman, H.J. (1994) Trophoblast infiltration. Reprod. Med. Rev., 3, 137–157.
Lawson, N.D. and Berliner, N. (1998) Representational difference analysis of
a committed myeloid progenitor cell line reveals evidence for bilineage
potential. Proc. Natl. Acad. Sci. USA, 95, 10129–10133.
Lawson, N.D., Khannagupta, A. and Berliner, N. (1998) Isolation and
characterization of the cDNA for mouse neutrophil collagenase —
demonstration of shared regulatory pathways for neutrophil secondary
granule protein gene expression. Blood, 91, 2517–2524.
Lerner, A., Clayton, L.K., Mizoguchi, E. et al. (1996) Cross-linking of T-cell
receptors on double-positive thyocytes induces a cytokine-mediated stromal
activation process linked to cell death. EMBO J., 15, 5876–5887.
Lisitsyn, N., Lisitsyn, N. and Wigler, M. (1993) Cloning the differences
between two complex genomes. Science, 259, 946–951.
Lopez, A.J. (1998) Alternative splicing of pre-mRNA: developmental
consequences and mechanisms of operation. Annu. Rev. Genet., 32, 279–305.
Maudelonde, T., Martinez, P., Brouillet, J.P. et al. (1990) Cathepsin-D in
human endometrium: induction by progesterone and potential value as a
tumor marker. J. Clin. Endocrinol. Metab., 70, 115–121.
Mignatti, P. and Rifkin, D.B. (1993) Biology and biochemistry of proteinases
in tumor invasion. Physiol. Rev., 73, 161–195.
Mori, K., Ogawa, Y., Tamura, N. et al. (1997) Molecular cloning of a novel
mouse aspartic protease-like protein that is expressed abundantly in the
kidney. FEBS Lett., 401, 218–222.
Niwa, H., Harrison, L.C. and DeAizpurua, H.J. (1997) Identification of
pancreatic beta-cell related genes by representational difference analysis.
Endocrinology, 138, 1419–1426.
Robertson, W.B., Khong, T.Y., Brosens, I. et al. (1986) The placental bed
biopsy: review from three European centers. Am. J. Obstet. Gynecol., 155,
401–412.
Sambrook, J., Fritsch, E.F. and Maniatis,T. (1989) Molecular Cloning — A
Laboratory Manual. Cold Spring Harbour Laboratory Press, New York.
Sowers, J.R., Eggana, P., Kowal, D.K. et al. (1993) Expression of renin and
angiotensinogen genes in preeclamptic and normal human placental tissue.
Hypertens. Preg., 12, 163–171.
Tang, J. (1979) Evolution in the structure and function of carboxyl proteases.
Mol. Cell. Biochem., 26, 93–109.
Tang, J, and Wong, R.N. (1987) Evolution in the structure and function of
aspartic proteases. J. Cell. Biochem., 33, 53–63.
Wada, J., Kumar, A., Ota, K. et al. (1997) Representational difference analysis
of cDNA of genes expressed in embryonic kidney. Kidney Int., 51,
1629–1638.
Welford, S.M., Gregg, J., Chen, E. et al. (1998) Detection of differentially
expressed genes in primar tissues using representational differences analysis
coupled to microarray hybridization. Nucl. Acids Res., 26, 3059–3065.
Zhou, Y., Fisher, S.J., Janatpour, M. et al. (1997a) Human cytotrophoblasts
adopt a vascular phenotype as they differentiate. A strategy for successful
endovascular invasion? J. Clin. Invest., 99, 2139–2151.
Zhou, Y., Damsky, C.H. and Fisher, S.J. (1997b) Preeclampsia is associated
with failure of human cytotrophoblasts to mimic a vascular adhesion
phenotype. One cause of defective endovascular invasion in this syndrome?
J. Clin. Invest., 99, 2152–64.
Received on April 30, 1999; accepted on July 16, 1999
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