Table Of ContentZOOLOGICAL SCIENCE 9: 337-342 (1992) © 1992Zoological SocietyofJapan
Changes in Timing and Site of Appearance of a Protease
in Xenopus Embryos
Shohei Miyata1 Yasuo Nishibe2 Michiko Sendai3 Isao Katayama2
, , , ,
Teruhiko Iino4 and Hirozi K. Kihara1
1Laboratory ofResearchfor Biosynthesis and Metabolism, Keio University, School of
Medicine, Tokyo, 160, 2First Department ofPathology, Saitama Medical School,
Saitama, 350-04 and Department of^Chemistry, and ^General Education,
College ofHumanities and Science, Nihon University, Tokyo 156, Japan
—
ABSTRACT A thiol protease was purified from embryos of Xenopus laevis. This protease has a
relativemass(Mr)of43k-44K. AntiserumraisedagainsttheproteasewasusedforanalysisbyWestern
blotting of proteins from embryos at various stages and from adult liver and heart. A band
M
corresponding to a protein with an r ofapproxmately44k was detected in the unfertilized eggs and
embryoswiththe monospecific antiserum. The size ofthe protein that reactedwiththe antiserum ina
preparationofproteinsfromtheliverofXenopuslaeviswasdifferentfromthatoftheproteinsfromthe
eggs and embryos (Mr, approximately 70k). Immunohistochemical localization with the antiserum
revealed that the protease was more abundant in the animal hemisphere and in the cells derived from
the animal hemisphere than in other cells of the embryo. The protease was highly enriched in the
cytoplasm of ectodermal cells than the cytoplasm of endodermal cell. When proteins from Xenopus
embryos were used as substrate, one protein having M of31 K was mainly digested.
r
INTRODUCTION MATERIALS AND METHODS
Animal cells contain many different proteases
and there are both lysosomal and non-lysosomal Assay ofproteolytic activity
pathways of protein degradation [1-3]. We have The proteolytic activity was determined with
purifiedanacidicproteasefromXenopusembryos. 3H]BSA as substrate and was assayed by measur-
[
Theproteaseissensitivetoantipain, leupeptinand ing the acid-soluble radioactivity released from
iodoacetic acid but it is insensitive to phenyl- [3H]BSA, as reported previously [4].
methylsulfonylfluoride andpepstatin [4]. Ithasan
M of 43 k-44k and can be dissociated into two Preparation of embryos and purification of the
subrunits with M of about 30k and 13 k. The protease
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proteolytic activity is activated by nucleic acid. Xenopus eggs were allowed to develop to the
The effect ofinhibitors, molecularweight, subunit tail-bud stage. The embryos were immersed in
structure, pH optimum, and phenomenon of cold acetone and homogenized in a glass
activationbynucleicacidseemtobedifferentfrom homogenizer. The homogenate wascentrifuged at
those of the proteases described thus far [1-3]. 3,000rpm for 10min at 4°C. The pellets were
The purpose of the present study was to monitor dehydrated by treatment with several changes of
temporal changes in the level, the distribution and acetone, dried under N2, and stored at —20°C.
the intracellular localization of the protease pro- The procedure for purification of the protease
tein in Xenopus eggs and embryos. has been described previously [4]. Twenty grams
of the acetone-dried embryos were homogenized
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Accepted December 24, 1991 in 200ml of 0.1 acetate buffer, pH5.0, which
Received October 26, 1991 contained 0.1% Triton X-100 and0.1 mM EDTA,
338 S. Miyata, Y. Nishibe et al.
in a glass homogenizer. The homogenate was then developed with a solution ofthe substrate for
centrifuged at 7,000Xg for 20min. The proteoly- peroxidase which consisted of azino-di (ethyl-
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tic activity in the supernatant was concentrated by benzthiazoline) sulfonic acid in 0.1 citrate buf-
fractionation with acetone (20-50%). The result- fer, pH4.2, supplemented with 0.03% hydrogen
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ing precipitate was dissolved in 0.1 acetate peroxide. The enzymatic reaction was terminated
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buffer, pH5.0, that contained 0.2 NaCl. The by washing with water. Proteins in gels were
extract was dialyzed against the same buffer and detected by silver staining [7].
succesively chromatographed on columns of
Sephadex G-75, CM-cellulose, and hydroxylapa- Light microscopy
tite. The final preparation ofenzyme represented Eggs and embryos were fixed in 2% paraform-
mM
a 16,500-fold purification. aldehydeand2.5% glutaraldehydein 10 phos-
phate buffer pH7.5, for4hrat4°C, washed thrice
Production ofantiserum against theprotease with PBS, dehydrated in a graded ethanol series,
New Zealand White rabbits were immunized cleared in 0.03% Nonidet p-40 in chloroform, and
withtheprotease. Onehundredmicrogramsofthe embeddedinTissuePrep (FischerScientific). Thin
protease, emulsified in Freund's complete ad- sections were cut at 4jum and deparaffinized with
juvant (GIBCO), were administered intradermal- xylene. Immunohistochemicalstainingwascarried
ly. Rabbits were given three booster injections out by modified version ofthe procedure ofWein-
intradermally with 100jug protease without ad- man et al. [8]. The sections were rinsed thrice in
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juvant. Bloodwascollected 1 weeklater. IgGwas PBS, and treated with 0.02 glycine in PBS and
purified from rabbit serum by chromatography on with 4% BSA in PBS. These were first labeled
columns ofDEAE-cellulose and Sephadex G-200, withantiserumagainsttheproteasediluted 1 :20in
withsubsequentprecipitaionwith33% ammonium 1% BSA in PBS, washed 4 times with PBS,
sulfate at 4°C. post-labeled with a biotin-labeled anti-rabbit IgG
diluted 1 :50in 1% BSAinPBS, andwashedthrice
Immunoprecipitation oftheprotease with PBS. The sections were reacted with avidin-
Various amounts of antiserum or control serum biotin-peroxidase complex, washed thrice with
were added to 150//l of PBS that contained PBS, reactedwith0.02% hydrogenperoxidase and
purifiedenzyme (0.22jug). Afterincubationfor48 diaminobenzidine tetrahydrochloride in PBS for
hr at 4°C, the mixture was centrifuged in a micro- visualization, and rinsed 4 times in distilled water.
fuge for 10min. The residual activity in the
supernatantwas determined with 3H]BSA as sub-
[ RESULTS
strate.
Western BlotAnalysis Immunoprecipitation oftheprotease
Proteins from embryos at various stages were To study the characteristics of the antiserum,
fractionated by electrophoresis on 15% polyacryl- immunoprecipitation of the protease was carried
amide gels [5] and then transferred to nitrocellu- out. The enzymatic activity remaining in the
lose membranes in a buffer that contained 62.5 supernatant was measured with 3H]BSA as sub-
mM Tris, 192mM glycine (pH8.7) and 20% strate after addition of aliquots[of antiserum or
methanol [6], with a current of 1.2mA/cm2 for90 control serum (Fig. 1). The addition ofincreasing
min at room temperature. The membranes were amounts of antiserum led to the gradual loss of
incubated with blot buffer (5% (mass/vol.) nonfat proteolytic activity, demonstrating that the anti-
dry milk in PBS) for 2hr and then with antiserum serum reacted with the active enzyme protein.
diluted 1 :200inblotbufferfor lhr. Afterwashing
with PBS, each membrane was exposed to anti- Screening with antiserum against the protease by
rabbit IgG conjugated with peroxidase in blot immunoblotting
buffer for 1 hr, washed three times with PBS, and Samplesofthe proteins from embryosatvarious
Protease in Xenopus Embryo 339
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100 o~
o
-67
K
<
—
ASK
LU
<
LU
Oh-
cr -25K
CL
20 40 60 120
IgG(yjg/0-2ml)
Fig. 1. Immunoprecipitaiton of the protease. Various
amountsofantiserumorcontrolserumwereaddedd
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to 0.01 phosphate-buffered saline that contained
purified enzyme. Enzymatic activity remaining in
the supernatant was measured after centrifugation, Fig. 2. Screening with antiserum for protease in
by measuring of acid-soluble radioactivity released embryos by immunoblotting.
from [3H]BSA. •. Antiserum; O, control serum. Purified enzyme protein was subjected to elec-
trophoresis in a 15% polyacrylamide gel and de-
tected by silver staining (Lane 1). Proteins (20/ig)
stages and from the liver of adult frogs were from unfertilized eggs, and from embryos at the
fractionated by gel electrophoresis, transferred to morula, gastrula, neurulaandtailbudstages (Lanes
nitrocellulose membranes, and subjected to im- 2-6) and proteins (20^g) from liver (lane 7) and
munoblotting with antiserum (Fig. 2). The ahea1rt5%(lapnoel8y)acorfyXleanmoidpeusglealeviasnwderterafnrsafcetriroendatteodian
purified enzyme gave a single band after elec- nitrocellulose membrane. Proteinwasvisualizedby
trophoresis in a 15% polyacrylamide gel (Fig. 2, treatment with antiserum, lane 2, unfertilized egg;
lane 1). The relative mass (Mr) ofthe enzyme was lane 3, embryo at morula stage; lane 4, embryo at
estimated to be about 44k. On screening with gastrulastage; lane 5, embryo atneurulastage; lane
antiserum of proteins from unfertilized eggs and 6;embryoattailbudstage;lane7,liverofXenopus;
lane 8, heart of Xenopus. Standard proteins used
from embryos atthe morula, gastrula, neurulMa and werebovineserumalbumin(67k), heneggalbumin
tail-bud stages, a band corresponding to an r of (45k) and chymotrypsinogen A (25k).
about 44k was visualized from embryos at all
stages (Fig. 2, lanes 2-6). When control serum embryos at the morula, gastrula, neurula and
was employed no bands were detected from tail-budstages. Theprotease in earlyembryoswas
embryos at any stage or from the adult liver (data already present as a maternal proteins in unfertil-
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not shown). The band corresponding to an of ized eggs.
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44k coincided in terms of size with the purified When a sample ofthe proteins from the liver of
protease detected by silver staining. The resulted Xenopuslaeviswasimmunoblotted, aproteinwith
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indicate that the antiserum was monospecific for r of 70k was visualized by treatment with anti-
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the protease protein from embryos. The relative serum, but the band corresponding to an of44
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intensity of the bands was almost constant among k was not observed (Fig. 2, lane 7). Also, the
the proteins from unfertilized eggs and from protein of 44k was not detected in the proteins
340 S. Miyata, Y. Nishibe et al.
from the heart by immunoblotting with the anti-
serum, while the protein of 70k was detected in Immunohistochemical analysis of the distribution
heart tissue (Fig. 2, lane 8). oftheProtease durin8 development
Thedistributionoftheproteasewasexaminedin
G :
i ; ... "\;x
•
' .": • • /"•.
A
. .
Fig. 3. Immunohistochemical localization ofthe protease in dissected eggs and embryos.
All sections were cut along the animal-vegetal axis and stained with antiserum (A-D, I andJ) orcontrol serum
(E-H). AandE,Unfertilizedegg;BandF,embryoatmorulastage;CandG,embryoatgastrulastage;DandH,
embryo atneurulastage; I, highermagnificationofembryo atgastrulastage;J, higermagnificationofembryoat
neurula stage.
Abbreviations are as follows: ap, animal pole; ec, ectoderm; en, endoderm; m, mesoderm; nt, notochord; s,
somites; vp, vegetal pole. Bar length: A-H, 0.25mm; I and J, 0.025mm.
Protease in Xenopus Embryo 341
sections of eggs and of embryos at various stages 1 2
by immunostaining with the antiserum. The pro-
teasewasdetectedasadarkdepositafterimmuno-
histochemicalstaining. Sectionswerecutalongthe
animal-vegetal axis. In the unfertilized egg, the
animal region showed strong immunoreactivityfor
the protease antigen (Fig. 3A). The animal region
atthemorulastagealsocontainedahigherconcen-
tration ofthe protease than did the vegetal region
(Fig. 3B). Immunohistochemical localization of
the protease antigen in the gastrula is shown in
Figure3C. The ectoderm was stained more
stronglythanthe endoderm. Intheembryosatthe
neurula stage, the intensity of the staining de-
creased from the ectoderm, through the Fig. 4. Digestion ofembryo proteins
lmaeyseordoefrmc,elltsootfhetheendeocdteordmer(mFiwg.as3Dm)o.stThsetroountgelry 0E.m1bMryoasceattattehebunfefuerrulaatsptHa5ge.0w.ereThheomohgoemnoigzeendatien
was centrifuged in a microfuge and the supernatant
stained and cells derived from the dorsal wasusedassubstrate. Theembryoproteins(40/ig)
mesoderm, such as the notochord and somites, were treated with the enzyme (0.5jug), in 0.1 M
also stained with higher intensity than the en- acetate buffer at pH3.8.
doderm. The animal portion of the egg develops
into ectodermal tissue, while an explant of the extent of digestion of the embryo proteins was
vegetal portion forms endodermal tissue. Furth- assessed by SDS-polyacrylamide gel elec-
ermore, when animal pole cells are put in contact trophoresis. When embryo proteinswere digested
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with vegetal pole cells, the fate ofthe animal pole in the 0.1 acetate buffer at pH3.8, a protein
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cells changes and they form mesoderm tissue [9, with r of 31 k was the most susceptible to diges-
10]. Thus, in our experiments, the ectoderm and tion (Fig. 4, lane 2) (Fig. 4, lane 1, enzyme-free).
mesoderm stain more heavity than the endoderm
as a result ofthe fact that the animal region stains
DISCUSSION
more heavily stronger than the vegetal region. In
sectionstreatedwith control serum, no differences The developmental profile of a novel thiol pro-
among the regions were detected (Fig. 3E-3H). tease in Xenopus was monitored using and anti-
To define the intracellurar localization of the serum raised against the protease. On screening
protease, magnified photographs ofsections at the with antiserum by immunoblotting, the protein
gastrulaandneurulastageswereexamined (Fig. 31 that reacted with the antiserum was detected
and J). The cytoplasm without yolk granules or amongthe proteins from the unfertilized egg. The
particles in the ectodermal cells at the gastrula concentration of protease was almost the same
stage was stained more darkly than the cytoplasm amongthe proteinsfrom unfertilizedeggs asitwas
in the endodermal cell (Fig. 31). The cytoplasm among proteins from embryos at the morula, gas-
without particles in the ectodermal cells at the trula, neurula and tail-bud stages. However, we
neurula stage was also stained as a dark deposit have already reported that the proteolytic activity
(Fig. 3J). But the endodermal cells were weakly is barely detectable in the unfertilized egg, when
stained. assays of thiol protease sensitive to antipain is
performed in the presence ofDTT [11]. Thus, the
Digestion ofembryoproteins proteolytic activity seems to be inhibited by some
The activity of the protease with embryo pro- unidentified mechanism in the unfertilized egg.
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teins as substrate is shown in Figure 4. Embryo Theproteasewith rof44kwasnotdetectedin
proteins were treated with the enzyme, then the adult tissues, and a protein of70kwas detected in
342 S. Miyata, Y. Nishibe et al.
these tissues. The protease with M of 44k 4 Miyata, S., Yoshida, Y and Kihara, H. K. (1989)
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appears to exist only in the embryo. Purification and characterizaiton ofa protease from
The distribution of the protease was studied in Xenopus embryos Eur. J. Biochem. 186: 49-54.
5 Laemmli, U. K. (1970) Cleavage of structural
embryos at various stages by immunohistochem-
proteinsduringtheassemblyoftheheadofbacterio-
ical methods. The protease was present at high phage T 4 Nature 227: 680-685.
levels in the animal region of the unfertilized egg 6 Towbin, H., Staehelin, T. and Gordon, J. (1979)
and at the morula stage, and in cells of the Electrophoretic transfer ofproteins from polyacryl-
ectodermal lineage in the early embryo. Further- amide gels to nitrocellulose sheets: Procedure and
more, the protease was mainly detected through- some applications. Proc. Natl. Acad. Sci. USA 76:
4350-4354.
out the cytoplasm without yolk granules or parti-
7 Morrissey, J. H. (1981) Silver stain for proteins in
cles in ectodermal cells atthe gastrula and neurula polyacrylamide gels: A modified procedure with
stage. It has been reported that oocytes and enhanced uniform sensitivity Anal. Biochem 117:
unfertilized eggs have lysosome-like organelles. 307-310.
These lysosomes appear to be located in a 8 Weinman, S., Ores-Carton, C. Rainteau, D. and
peripheral zone of the cytoplasm [12, 13]. Thus, lPoucsazlkiizna,tioSn.of(1c9a8l6m)oduIlmimnuannodelpehcotsrphoonlimpiacsreosAco2piicn
this protease seems not to be localized in the spermatozoa 1. J Histochem. Cytochem. 34: 1171—
lysosome. 1179.
Many non-lysosomal proteases have been 9 Nieuwkoop, P. D. (1969) The formation of
purified and characterized [1-3]. We reported mesoderm in urodelan amphibians. 1. Induction by
previously that the proteolytic activity which is theendoderm. Wilheim Roux' Arch. 162: 341-374.
10 Nakamura, O, Takasaki, H. and Mizohata, T.
sensitive to the protease inhibition, antipain in-
(1970) Differentiation during cleavage in Xenopus
creases during early development and antipain
laevis. Acquision of self-differentiation capacity of
inhibits the incorporation of [3H] uridine into the dorsal marginal zone. 1. Proc. Japan Acad. 46:
RANinXenopusembryos [11, 14]. Thisstudywas 694-699.
carried out by antiserum raised against the anti- 11 Miyata, S. and Kihara, H. K. (1988) Charactariza-
pain sensitive protease. Following fertilization, tion ofproteolytic activities in embryos ofXenopus
DNA laevis. Comp. Biochem. Physiol. 91B: 651-656.
the synthesisofproteins, replication andcell
divisionbegin [15, 16]. SynthesisofRNAbecomes 12 yBoulsksopnr-oMtaebiinlsltoot,lysS.oso(m1e9s84i)nthEenvdioteslolmogeesnictroaoncsyfteer
detectable at the midblastula stage [16-18]. This ofthe trout. Biol. Cell. 51: 53-66.
protease may function as one of components re- 13 Wall, D. A. and Meleka, I. (1985) An unusual
quiredformaintenance ofhigh rates ofthe synthe- lysosome compartment involved in vitellogenin en-
sisofRNA, proteins orDNA in the early embryo. docytosis by Xenopus oocytes. J. Cell Biol. 101:
1651-1664.
14 Miyata, S., Shimazaki, T., Okamoto, Y., Motegi,
ACKNOWLEDGMENTS N., Kitagawa, M. and Kihara, H. K. (1988) Inhibi-
tion ofRNA synthesis in embryo ofXenopus laevis
We
are grateful for the skillful technical assistance in by protease inhibitor. J. Exp. Zool. 246: 150-155.
light microscopy of Mr. Kouji Ootomo of Saitama 15 Ecker, R. E. and Smith, L. D. (1971) The nature
Medical School. Our thanks are also due to Mr. Hideo and fate of Rana pipiens proteins synthesis during
Kanei ofKeio University for his able assistance. maturaiton and early cleavage. Dev. Biol. 24: 559-
576.
REFERENCES 16 Gurdon,J. B. (1974) Thecontrolofgeneexpression
in animal development. Clarendon Press, Oxford.
1 Hershko, A and Ciechanover, A. (1982) Mecha- 17 Shiokawa, K., Misumi, Y. and Yamana, K. (1981)
nismsofintracellularproteinbreakdownAnn. Rev. Demonstration of rRNA synthesis in pre-gastular
Biochem. 51: 335-364. embryo of Xenopus laevis. Develop., Growth and
2 Pontremoli, S. and Melloni, E. (1986) Extralyso- Differ. 23: 579-587.
somal protein degradation Ann. Rev. Biochem. 55: 18 Newport, J. and Kirschner, M. (1982) A major
455-481. developmentaltransitioninearlyXenopusembryos:
3 Bond, J. S. and Butler, P. E. (1987) Intracellular 1. Characterization andtimingofcellularchangesat
proteases Ann. Rev. Biochem. 56: 333-364. the midblastura stage Cell. 30: 675-686.
