1 Division of Developmental Biology, Cincinnati Children's Hospital Research
Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
2 Department of Cell and Developmental Biology, Oregon Health and Science
University, Portland, OR 97201, USA
3 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
Cambridge CB10 1SA, UK
4 Wellcome Trust/Cancer Research Gurdon UK Institute, University of Cambridge,
Tennis Court Road, Cambridge CB2 1QR, UK
* Author for correspondence (e-mail: heabq9{at}chmcc.org)
Accepted 29 November 2004
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SUMMARY |
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Key words: PACE4, TGFß, Mesoderm induction, Vg1
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Introduction |
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XPACE4 is a secreted, heparin-binding member of the pro-protein convertase
(PC) family (Mains et al.,
1997; Tsuji et al.,
2003
). It recognizes the multibasic consensus motif, RXXR, and
cleaves substrates, including TGFß family members, to release the active
mature protein. PCs are important in vertebrate embryogenesis (for a review,
see Taylor et al., 2003
). In
vertebrate embryos, they have been shown to process activin
(Roebroek et al., 1993
),
TGFß1 (Dubois et al.,
1995
), BMPs (Cui et al.,
1998
) and Nodal (Constam and
Robertson, 1999
; Beck et al.,
2002
; Constam and Robertson,
2000
). PCs have also been implicated in remodeling of the
extracellular matrix by processing matrix metalloproteases
(Leighton and Kadler, 2003
;
Yana and Weiss, 2000
) and
regulating cell adhesion by processing integrins
(Berthet et al., 2000
;
Stawowy et al., 2004
).
Recently glypican 3, a heparan sulfate proteoglycan involved in morphogen
gradient formation was found to be processed by PCs
(De Cat et al., 2003
).
In this study, we have analyzed the role of XPACE4 in mes-endoderm specification in Xenopus. We find that XPACE4 maternal mRNA is localized to the vegetal hemisphere of oocytes, whereas zygotic mRNA is localized to the notochord, the brain and a subset of endodermal precursors. We show that XPACE4 protein is essential for normal development in vivo, and for the production of endogenous mesoderm inducing signals. By comparing the cleavage of overexpressed tagged TGFß proteins in wild-type and XPACE4-depleted backgrounds, we identify the TGFß proteins that are specific substrates for cleavage by XPACE4.
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Materials and methods |
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Oligos and mRNAs
The antisense oligonucleotide complementary to XPACE4 used in this
study (AS-5) was
5'-C*A*A*GGTTCAGGTAGCC*G*T*-3',
where residues with phosphorothioate bonds are indicated by an asterisk
(*) and was used in doses of 4-5 ng. XPACE4 morpholino
oligo was 5'-GCATGTTTGAAATGCTCAGAGGGAG-3' and was used in doses of
30-45 ng.
To generate HA-tagged TGFßs, one HA epitope (YPYDVPDA) was inserted at the C terminus immediately after the coding sequence, and before the stop codon by high fidelity PCR. The inserts were subcloned into pCS2+ vector. Each construct was then sequenced for confirmation and tested for activity to ensure that the activity of the protein was not disrupted by the presence of the tag. The linearized plasmids were purified and mRNA was transcribed with SP6 polymerase using the Megascript kit (Ambion). The doses and sites of injection are described in the text.
Real-time PCR
Total RNA and cDNA were prepared according to Zhang et al.
(Zhang et al., 1998). cDNA was
synthesized using oligo dT primers, or random hexamer (R6) primers where
indicated. Real-time RT-PCR was carried out using the Light Cycler System
(Roche) as described by Kofron et al.
(Kofron et al., 2001
) using
the primers and cycling conditions as listed in
Table 1. All samples were
normalized to levels of ornithine decarboxylase (ODC), which was used
as the loading control. Every experiment was repeated at least twice with
different oocyte and embryo batches to show that the results were
reproducible.
|
Whole-mount in situ hybridization
Oocytes at all stages were removed from follicle cells by incubating in 2
mg/ml collagenase in Ca2+ and Mg2+-free 1xMMR and
fixed in MEMFA for 1-2 hours. To increase probe penetration into the yolky
vegetal hemisphere, fixed oocytes and embryos were bisected into halves in the
animal-vegetal axis using a clean scalpel blade, refixed in MEMFA, washed in
PBS and transferred into 100% methanol. Whole-mount in situ protocol was
modified from Harland (Harland,
1991). XPACE4 antisense probe was transcribed using T7
polymerase after pCS2+XPACE4 vector was digested with EcoRI.
For the sense probe, pCS2+ XPACE4 vector was digested with
XhoI and transcribed using SP6 polymerase. In situs were developed
using BM Purple as substrate (Roche).
Nieuwkoop assay
Vegetal masses were dissected from control and XPACE4-depleted
embryos at stage 9. Wild-type animal caps from stage 8-9 embryos were
co-cultured with vegetal masses for 1-2 hours. They were then separated
carefully using tungsten needles and contaminating vegetal cells, recognized
by their vital dye coloring, were removed. The caps were incubated until they
reached the desired stage and were frozen down for analysis.
Paracrine assay
The paracrine assay was carried out as described previously
(Lustig and Kirschner, 1995).
Oocytes were injected with an antisense oligo, cultured for 48 hours at
18°C, and then injected vegetally with 150-200 pg of Xnr1 mRNA,
before culturing for an additional 12-24 hours to allow time for protein
synthesis and processing. The oocytes were transferred into wells in agar
plates with indentations, and stage 9 animal caps were placed on their animal
poles, bringing the inner cell layer of caps into contact with the surface of
the oocytes. Caps were co-cultured with oocytes in OCM for 1 hour at room
temperature, and then carefully separated from the oocytes. The caps were
cultured separately until sibling embryos reached the desired stage when they
were frozen down for analysis.
Luciferase assay
Control and XPACE4-depleted embryos were injected vegetally at the
two-cell stage with 50 pg of the firefly luciferase reporter construct
pGL3-ARE-luciferase described previously
(Huang et al., 1995) together
with 20 pg of control HSTK Renilla luciferase plasmid. Batches of
four embryos were collected in triplicate for each injection mixture at stage
10.5. Luciferase assays were performed using the Dual Luciferase Reporter
Assay system (Promega) as described
(Kofron et al., 2004
). The
assay was repeated twice, and a representative experiment is shown.
Blastocoel fluid withdrawal
Needles were prepared in the size range of 10-20 nl/second, and rinsed with
the protease inhibitor PIC (Roche). The needle was inserted through the
blastocoel roof into the blastocoel cavity of early gastrula stage embryos,
and the blastocoel fluid was withdrawn gradually, using a Medical Systems
picoinjector. Care was taken not to contaminate the fluid with cellular
debris. Depending on the batch of embryos, 0.1-0.2 µl of fluid/embryo was
collected. After blastocoel fluid from 10 embryos was collected, the fluid was
snap frozen. Similar volumes of fluid were withdrawn from control and
experimental embryos.
Western blots
Protein extracts were prepared from batches of 3-5 embryos using 10 µl
homogenization buffer/embryo. Homogenization buffer for anti-HA blots is 2.5%
IGEPAL CA-630 Sigma I3021, 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1%
Triton X-100, 0.1% SDS; containing 1:100 PIC (Sigma P-8340). For
anti-phospho-SMAD2 blots, homogenization buffer is 20 mM Tris pH 8.0, 2 mM
EDTA, 5 mM EGTA, 0.5% NP-40 (Sigma), 25 mM sodium ß-glycerophosphate, 100
mM NaF, 20 nM Calyculin A, 10 mM sodium pyrophosphate, containing 1:100 PIC
and PMSF. Homogenates were spun at 13,000 g for 10 minutes and
the supernatant was collected and mixed with equal volume of 2x reducing
sample buffer. Blastocoel fluid samples were also spun down and 10 µl of
sample buffer was added. Samples were boiled and 0.5-1 embryo equivalents of
protein extracts or the entire blastocoel fluid samples were loaded on 10-12%
SDS-PAGE gels and transferred on to nitrocellulose membranes. The membranes
were blocked with 5% NFDM in PBS-Tween overnight at 4°C for
anti-phospho-Smad2 blots and 1-2 hours at room temperature for others.
Anti-phospho-Smad2 antibody (Cell Signaling Technology #3101) and anti-Smad2
antibody (BD Transduction Laboratories) were used at a dilution of 1:500.
Anti-HA high affinity rat monoclonal antibody 3F10 (Roche 1-867-423) was used
at a dilution of 1:1000. Signal detection was carried out using the Amersham
ECL detection system. No signal was detected with this HA antibody in the
uninjected control samples. The relative mobility of each protein was
calculated by comparison with a standard curve generated by migration of the
prestained kaleidoscope standards (BioRad). One HA tag is approximately 1.1
kDa.
As a loading control, membranes were stripped after signal detection, and
incubated with -tubulin antibody (DM1A, Neomarkers) at 1:10,000. No
-tubulin was detected in the blastocoel fluid samples. The experiments
were repeated at least twice.
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Results |
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These results show that maternal XPACE4 mRNA is localized to the vegetal hemisphere of the oocyte and is regulated by polyadenylation, while during embryogenesis, it is localized in endodermal precursors and later in specific areas of all three germ layers.
Depletion of maternal XPACE4 mRNA results in reduced enzymatic function in paracrine assays
Antisense oligos designed against the XPACE4 coding sequence were
tested for efficiency by injection into the vegetal poles of full-grown
oocytes, which were incubated for 48 hours before real-time RT PCR analysis
for XPACE4 mRNA depletion. Fig.
3A shows that antisense oligo 5 (AS-5) is the most efficient, and
depletes XPACE4 mRNA to 5-10% of control levels. To increase the
stability of oligo, phosphorothioate modified AS-5 (AS-5MP) was used.
Fig. 3B shows that the oligo
does not affect mRNA levels of a related maternal convertase, furin
(XFurA).
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As mouse Nodal is a known substrate of mammalian PACE4
(Beck et al., 2002), we
assessed the loss of activity of XPACE4 in a functional assay. For paracrine
assays, control and XPACE4-depleted oocytes were injected with
Xnr1 mRNA and co-cultured with wild-type animal caps as described in
the Materials and methods. Fig.
3D shows that the depletion of XPACE4 decreases the
inducing activity of Xnr1. Although the organizer genes, chordin
(Chd) and goosecoid (Gsc), and the mesodermal gene
Xbra and endodermal gene XSox17
are induced in animal
caps co-cultured with control oocytes overexpressing Xnr1 mRNA, caps
cultured with XPACE4-depleted oocytes show little inducing activity.
This indicates that the Xnr1-processing activity of endogenous XPACE4 protein
is reduced by the antisense oligo-mediated depletion of the maternal
XPACE4 mRNA.
XPACE4 is required for mesoderm induction
To determine the function of maternal XPACE4 during embryogenesis, we used
the host transfer technique to fertilize XPACE4-depleted oocytes.
XPACE4-depleted embryos develop normally through the cleavage and
blastula stages. The phenotype caused by maternal XPACE4 depletion is
obvious at gastrulation, when the formation of the blastopore is delayed
compared with controls (Table
2). This phenotype is rescued by the reintroduction of
XPACE4 mRNA into sibling XPACE4-depleted embryos
(Fig. 4A,B). After 1-2 hours
delay, the blastopore ring forms and closes and the embryos continue to
develop, with varying degrees of abnormality of anterior structures
(Fig. 4C;
Table 3). Similar phenotypes
are observed when 30-45 ng of XPACE4 morpholino oligo is used (data
not shown).
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First, equatorial regions, which are specified at the late blastula stage to form mesodermal tissue, were dissected from control and XPACE4-depleted embryos, cultured until the neurula stage and analyzed for the expression of mesodermal (MyoD and cardiac actin) and neural (NCAM) markers. Fig. 4D shows that control equatorial explants undergo the typical convergent extension movements indicative of mesoderm induction. By contrast, XPACE4-depleted equatorial explants are unable to undergo convergent extension movements, a deficiency that is specific to XPACE4-depletion as it is rescued by the reintroduction of XPACE4 mRNA. The expression of both cardiac actin and MyoD expression is reduced by XPACE4-depletion and is rescued by the reintroduction of XPACE4 mRNA (Fig. 4E). Neural tissue is normally secondarily induced in control equatorial explants. Neural induction assayed by the expression levels of NCAM is reduced by XPACE4-depletion and rescued by XPACE4 mRNA. Marker analysis of gastrulae using real-time RT-PCR confirms that the expression of mes-endodermal genes is reduced in XPACE4-depleted embryos. In particular, the general mesodermal gene Xbra, and the organizer gene chordin (Chd) are reproducibly reduced in expression, while Xnr1, which is known to be positively autoregulated, and Xnr3 are also affected. The expression of Chd, Xnr1 and Xnr3 is partially rescued by the reintroduction of XPACE4 mRNA (Fig. 4F). The expression of the mesodermal markers Fgf8 and derrière in whole embryos is little affected by XPACE4-depletion (data not shown).
Second, we asked whether endogenous mesoderm induction by vegetal masses
was reduced in XPACE4-depleted embryos, using Nieuwkoop assays
(Fig. 5A). Animal caps were
dissected from wild type mid-blastula stage embryos and co-cultured for 1-2
hours with vegetal masses dissected from control or XPACE4-depleted
embryos at the mid-blastula stage. After the induction period, the caps were
separated and cultured until the mid-gastrula stage and analyzed for induced
gene expression. Fig. 5A shows
that the mes-endodermal genes chordin (Chd),
goosecoid (Gsc), Fgf8, Xbra and
XSox17, which are targets of TGFß signaling, are
significantly induced in animal caps by wild-type vegetal masses, but not
XPACE4-depleted vegetal masses. This experiment was repeated twice
with similar results.
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Fourth, we determined the phospho-Smad2 levels in XPACE4-depleted embryos by western blots using a phospho-Smad2 specific antibody. Fig. 5C shows that XPACE4-depleted embryos have decreased phospho-Smad2 levels most significantly at the late blastula.
These results indicate that XPACE4 regulates endogenous mesoderm induction.
XPACE4 regulates processing of specific TGFß proteins during Xenopus embryogenesis
To determine which TGFß family members expressed during early
Xenopus embryogenesis are substrates for XPACE4, we assayed control
and XPACE4-depleted embryos for processing of different TGFß
proteins. We injected mRNA coding for HA-tagged proteins into the vegetal
cytoplasm of wild-type and XPACE4-depleted embryos at the two-cell
stage, and assayed for precursor and mature forms of proteins by western
blotting using a high-affinity HA antibody at the early gastrula stage. The HA
tag was inserted into the C terminus of each construct, allowing the
visualization of the mature protein, precursor protein and any stable
C-terminal-containing intermediaries of Xnr2, Xnr1, Xnr3, Xnr5, Vg1, ActivinB
and Derrière. In many cases, the analysis of whole embryo homogenates
showed that only a small percentage of the overexpressed proteins were
cleaved, making comparison between control and XPACE4-depleted
embryos difficult. Therefore, we took advantage of the fact that both
precursor and cleaved forms of the HA-tagged TGFß proteins are secreted
into the blastocoel cavity (Williams et
al., 2004). As this concentration of pro-protein and mature
protein is uncontaminated with cellular debris, it provides a more sensitive
assay for the determination of changes due to XPACE4-depletion. The
experiments were repeated at least three times and a representative experiment
is presented for each of TGFß protein
(Fig. 6A-H).
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These results demonstrate that endogenous XPACE4 cleaves exogenous Xnr1, Xnr2, Xnr3 and Vg1, but not ActivinB, Derrière and Xnr5. Taken together, this work provides the first evidence that XPACE4 is an essential vegetally localized regulator of mesoderm induction in Xenopus embryos, with specificity towards the TGFßs Xnr1, Xnr2, Xnr3 and Vg1.
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Discussion |
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XPACE4 and its mammalian ortholog are localized mRNAs
At the late blastula and early gastrula stages, the expression domains of
XPACE4 and several TGFß mRNAs, including Vg1, Xnr1 and Xnr2,
coincide. In mouse, processing depends on the ability of the secreted
pro-protein precursor and the PACE4/SPC4 enzyme to contact each other in the
extracellular space (Beck et al.,
2002; Constam and Robertson,
1999
). We have no direct evidence that endogenous XPACE4 is a
secreted protein, as specific antibodies are not available. However, we find
that uncleaved as well as cleaved forms of exogenous Vg1, Xnrs, ActivinB and
Derrière accumulate in a stable fashion in blastocoel fluid, and that,
for Xnr1, Xnr2 and Xnr3, the uncleaved pro-proteins accumulate more when
XPACE4 is depleted. The simplest explanation for this is that XPACE4 is
secreted. Thus, the pro-proteins are normally cleaved extracellularly by
secreted XPACE4, and accumulate more in the blastocoel in XPACE-depleted
embryos, because the enzyme is not there to process it. However, this remains
to be confirmed directly. Another possibility is that cleavage of endogenous
TGFßs occurs intracellularly, but that excess uncleaved and intermediate
forms are produced and secreted in blastocoel fluid as a result of the
excessive loading of the cells with these mRNAs (100-600 pg of mRNA for
HA-tagged TGFßs was injected). It will be important to determine whether
endogenous XPACE4 is present in blastocoel fluid when antibodies become
available.
Later in development, XPACE4 mRNA is found in the notochord, the
brain and the olfactory bulb, where its position correlates with expression of
rodent PACE4 in the developing CNS
(Akamatsu et al., 1997;
Zheng et al., 1997
), and in a
subset of cells in the endoderm correlating with the position of primordial
germ cells. Possible roles of XPACE4 in these locations remain to be
determined.
XPACE4 is required for mesoderm induction
The regulation of TGFß signaling in vertebrate embryogenesis is
crucial for correct tissue formation, and this study together with work in
mouse embryos shows the importance of subtilisin-family proteases in this
process (Beck et al., 2002;
Constam and Robertson, 1999
;
Constam and Robertson, 2000
).
Although the targeted loss of function of PACE4 in mouse embryos showed
relatively mild effects [causing only 25% of animals to die prenatally with
cardiac malformations, laterality defects and craniofacial abnormalities, and
75% to survive to birth (Constam and
Robertson, 2000
)], PACE4/furin double mutants showed severe
anterior visceral endoderm and axis formation defects, indicative of
disruption of Nodal cleavage and function
(Beck et al., 2002
). In the
studies presented here, we find that the predominant contribution of
XPACE4 mRNA is from maternal stores accumulated during oogenesis,
with relatively small and localized areas of synthesis of zygotic XPACE4
transcript. Two observations argue that the maternal store of protein
accumulated specifically in vegetal, endomesoderm precursors is stable and
long lived. First, the XPACE4 phenotype, delayed gastrulation and headless
development, was more pronounced the longer the oligo-injected oocytes were
incubated before fertilization (48 hours versus 24 hours). Longer incubation
after oligo injection would be expected to produce greater turnover of the
stable XPACE4 protein pool synthesized during oogenesis. Second, we found that
a morpholino oligo against XPACE4 was effective in causing the same phenotype
as AS-5MP oligo if the morpholino oligo was injected 2 days before
fertilization, but was not effective when injected into fertilized eggs (data
not shown). It will be interesting to determine if there is also a maternal
contribution of PACE4/SPC4 protein in mouse development, which allows the
relatively normal early development of mPACE4-/- embryos.
Several lines of evidence presented show the importance of XPACE4 in
mesoderm induction. First, loss of XPACE4 causes a loss of mesodermal markers
in whole embryos. Second, Niewkoop assays show that XPACE-depleted vegetal
masses are unable to induce wild-type animal caps to form mesoderm. Third,
loss of XPACE4 causes loss of ARE-luciferase activity concomitant with loss of
mesodermal gene expression. Last, phospho-Smad2, the immediate downstream
target of activin-type TGFß signaling, is also reduced in XPACE4-depleted
embryos. All approaches show the reduction of Nodal signaling activity due to
the depletion of maternal XPACE4. Since, of the TGFßs tested, we found
that Xnr1, Xnr2, Xnr3 and Vg1 are substrates for XPACE4 cleavage, it is likely
that the defect in mesodermal induction due to XPACE4 depletion is caused by
the reduction in mature forms of these proteins. Previously, we have shown
that Xnr3 is required for convergence extension movements and head formation
(Yokota et al., 2003), and
that Xnr1 and Xnr2 are able to rescue axis formation and mesoderm induction in
embryos depleted of TGFß signaling by antisense ablation of maternal VegT
(Kofron et al., 1999
;
Xanthos et al., 2001
;
Zhang et al., 1998
). We find
here that the Vg1-like protein Derrière is not a substrate for XPACE4.
Derrière is known to be important in establishment of tail and
posterior axial structures, and rescues VegT-depleted embryos to the extent of
causing late gastrulation and the formation of tails and dorsal axes but not
heads (Kofron et al., 1999
).
Thus, one explanation for the late gastrulation and posterior axial
development of XPACE4-depleted embryos is that normal Derrière function
is maintained. This hypothesis is supported by the fact that phospho-Smad2
levels approach control levels by mid-gastrulation when Derrière is
expressed (Fig. 5C). It will be
interesting to determine whether Derrière, ActivinB and Xnr5 are
substrates for the other maternally supplied PCs.
Comparisons of cleavage spectra and activities of HA-tagged TGFß proteins
To date, this work provides the most comprehensive comparison of the
cleavage spectra of seven TGFß proteins in embryo lysates and blastocoel
fluids, and the effect of depletion of one of the regulating enzymes on those
patterns. As the HA tags were placed at the C termini of the proteins, N
terminal pro-domain fragments were not visualized.
Table 4 correlates these
findings with the known biological activity of each of the proteins as
measured by mesoderm induction. Several conclusions can be drawn from the
comparisons.
First, XPACE4 regulates Xnr1, Xnr2, Xnr 3 and Vg1 but not Xnr5,
Derrière and ActivinB. As all of these proteins (with the exception of
ActivinB) share an RXXR consensus cleavage motif, the substrate specificity of
XPACE4 may be due to different residues flanking this consensus site.
Specificity and optimum activity of convertases have been documented to be
dependent on these flanking sequences
(Apletalina et al., 1998;
Henrich et al., 2003
), and
studies on Vg1 cleavage indicate that the presentation of the cleavage site in
a particular context and/or conformation is crucial for regulating cleavage
(Dohrmann et al., 1996
;
Thomsen and Melton, 1993
).
Second, we show here that the mature form of Xenopus Vg1 can be
detected by western blots using a high-affinity HA antibody, that this form
has weak mesoderm-inducing activity and that the processing of Vg1 is reduced
in XPACE4-depleted embryos. Previously, although native Vg1 protein
has been shown to be present vegetally in oocytes and early Xenopus
embryos (Dale et al., 1989),
the mature form was neither detected nor active in mesoderm induction
(Tannahill and Melton, 1989
).
In addition, while Vg1 chimeras with prodomains of either Activin or BMP
proteins were processed efficiently and were potent mesoderm inducers
(Dale et al., 1993
;
Thomsen and Melton, 1993
),
mature exogenous Vg1 could be detected but had no mesoderm-inducing activity
(Kramer and Yost, 2002
). By
contrast, the zebrafish homolog of Vg1, zDVR-1
(Dohrmann et al., 1996
) and
chick Vg1 (Shah et al., 1997
)
were detectable as mature proteins with weak mesoderm-inducing activity in
Xenopus oocytes.
The Vg1 used here has some sequence differences compared with the published
Vg1 sequence. One difference is a C-to-T transition resulting in a serine
residue (TCN codon) instead of a proline (CCN codon) at position 20. The
analysis of available EST sequences has suggested that both of these alleles
are present in Xenopus oocytes. The proline residue in the helix rich
N terminal region of Vg1 may be responsible for disruption of the structure,
and affect processing and render the protein functionless. This may explain
the previous observation that N-terminal signal peptide of Vg1 does not get
cleaved and Vg1 protein fails to dimerize
(Dale et al., 1993), whereas
the Vg1 with a serine residue at position 20 has weak mesoderm-inducing
activity (data not shown). It will be interesting to determine the function of
this Vg1 allele in detail and to understand the structural distinctions
between the different forms.
Third, the Xnr2 cleavage spectrum is more complex than that of the other
six proteins. Although the intermediate form of 33 kDa may be a degradation
product or caused by nonspecific cleavage at an upstream cryptic site, the
fact that it accumulates in an XPACE-depleted background suggests that it may
be formed by the specific pro-protein digestion activity of another
endoprotease, generating a processing intermediate that is subsequently
cleaved at the known downstream site. We have shown previously that BMP4
processing involves sequential cleavage that is important in regulating the
stability and activity of the mature protein
(Cui et al., 2001). Analysis of
Xnr2 protein sequence reveals the presence of a consensus RXXR motif located
70 amino acids upstream of the known cleavage site that would be
predicted to yield a protein form of
33 kDa upon endoproteolysis. Xnr2
generated from a precursor in which this upstream site is mutated, however,
maintains reduced activity, suggesting that processing at the downstream site
occurs independent of the upstream site and is sufficient for Xbra induction
activity. A double cleavage mutant of Xnr2 that fails to generate any mature
protein also induces Xbra, suggesting that unprocessed Xnr2 has some signaling
activity (Eimon and Harland,
2002
).
Fourth, as has been shown previously for Activin and Vg1, there is no simple correlation between the amount of mature form of each of the TGFßs and their biological activity. Here, Xnr5 and Activin are the most active proteins. They work at very low doses of RNA in mesoderm induction assays and yet accumulate less well than Xnr1 and 2 in whole embryo lysates. They do, however, accumulate substantially in blastocoel fluid. Cleaved forms of Xnr3 and Vg1 are only very weakly detected in either the lysates or the blastocoel fluid. Undoubtedly, the regulation of each TGFß is complex and individual, involving the interplay of stabilizing or competing intermediary cleavage forms, other competing co-expressed TGFß family members and the presence or absence of localized co-receptors. Specific loss-of-function and mutation analysis for each individual TGFß and detailed functional analysis of the other endoproteolytic enzymes are required to fully understand their function.
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ACKNOWLEDGMENTS |
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