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First published online 19 December 2007
doi: 10.1242/dev.015503
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Department of Molecular Biology, Cellular Biology, and Biochemistry, Box G-L173, Brown University, Providence, RI 02912, USA.
* Author for correspondence (e-mail: rhet{at}brown.edu)
Accepted 26 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Dityrosine, Peroxidase, Permeability, Fertilization envelope, Hydrogen peroxide
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wong and Wessel, 2006a). In
general, modifications of this matrix stabilize the protein barrier so that
digestion by a `hatching enzyme' is necessary before the embryo may escape to
begin feeding (Barrett et al.,
1971; D'Aniello et al.,
1997; Fan and Katagiri,
2001; Katagiri et al.,
1997; Kitamura and Katagiri,
1998; Lee et al.,
1994; Lepage and Gache,
1990; Mozingo et al.,
1993; Nomura et al.,
1997; Sawada et al.,
1990; Yamagami et al.,
1992). The timing of hatching varies among animals, occurring at
blastula for feeding embryos such as echinoderms, ascidians and mammals, or at
birth for embryos whose store of yolk sustains them throughout development,
such as teleosts, birds and reptiles.
The microenvironment enclosed upon creation of this zygotic extracellular
matrix is established by at least two egg contributions. First, hydration of
proteoglycans from egg vesicles establishes an aqueous cushion within the
perivitelline space, the volume that distances the embryo from the barrier
(Kay and Shapiro, 1985;
Larabell and Chandler, 1991;
Schuel et al., 1974;
Shapiro et al., 1989;
Talbot and Dandekar, 2003;
Talbot and Goudeau, 1988).
Second, the matrix itself is transformed by enzymes derived from the cortical
granules, a process that alters the matrix characteristics to resist
mechanical distortion and chemical dissolution (reviewed by
Shapiro et al., 1989;
Wong and Wessel, 2006a). In
anurans, a zinc metalloprotease is responsible for the physicochemical
alteration of their vitelline envelope
(Lindsay and Hedrick, 2004); a
functional homolog modifies the same target in the mammalian zona pellucida
(Bauskin et al., 1999;
Moller and Wassarman, 1989).
Conversely, inter-protein crosslinks are essential for the change in
extracellular matrix properties in insects
(Li et al., 1996), teleosts
(Chang et al., 2002;
Kudo, 1988;
Oppen-Berntsen et al., 1990)
and echinoderms (Foerder and Shapiro,
1977; Hall, 1978;
Wong et al., 2004). One or
many of these biochemical modifications are hypothesized to establish a
physical barrier separating the outside from the perivitelline space, thereby
maintaining a sterile, or even a dessicant-resistant, microenvironment
(Grey et al., 1974;
Kay and Shapiro, 1985;
Shapiro et al., 1989).
Two types of protein crosslinks are generally involved in the
transformation of the egg extracellular matrix
(Wong and Wessel, 2006a).
Transglutaminase establishes covalent epsilon (gamma-glutamyl)lysine bonds in
a calcium-dependent fashion in teleosts
(Chang et al., 2002;
Kudo, 1988;
Oppen-Berntsen et al., 1990)
and sea urchins (Battaglia and Shapiro,
1988). Peroxidase, however, forms dityrosine crosslinks via a
free-radical mechanism (Davies,
1987; Davies and Delsignore,
1987; Davies et al.,
1987; Gross, 1959;
Heinecke et al., 1993a;
Heinecke et al., 1993b;
Yip, 1966) in insects
(Li et al., 1996) and sea
urchins (Foerder and Shapiro,
1977; Hall, 1978).
The possible targets of both enzymes in the sea urchin fertilization envelope
are well defined, and include rendezvin, an alternatively spliced gene whose
vitelline layer (RDZ120) and cortical granule isoforms
(RDZ40, RDZ60, RDZ70, RDZ90) are
rich in CUB domains, as well as SFE1, SFE9, and proteoliaisin, which are each
enriched with tandem low-density lipoprotein receptor type A repeats (LDLrA)
(Wong and Wessel, 2004;
Wong and Wessel, 2006a). These
proteins and their various domains represent a diverse population with which
to test the target specificity of the crosslinking mechanisms. Here, we
identify a mechanism whereby specific protein crosslinking within the sea
urchin fertilization envelope establishes the limited permeability of this
filtration barrier (Kay and Shapiro,
1985).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Permeability assays
Fertilization envelope permeability was tested by measuring the diffusion
of fluorophore-conjugated dextrans into the perivitelline space. As
appropriate, eggs were dejellied in ASW acidified with HCl (pH 5.2) (S.
purpuratus) or calcium-free seawater (CFSW; L. variegatus) for
10 minutes, then washed three times in normal ASW (pH 8.0) to equilibrate the
pH. Fertilization without extracellular calcium was conducted in CFSW after
washing eggs three times in the respective medium to equilibrate the cells.
Sperm diluted into ASW containing egg jelly were used to inseminate eggs in
ASW, CFSW, or 1 mM 3-aminotriazole (3-AT; Sigma-Aldrich, St Louis, MO) in ASW;
eggs were then incubated for 20 minutes. Zygotes were transferred to Kiehart
chambers (Kiehart, 1982)
containing 5 µM of both anionic Cascade Blue dextran (3000 daltons) and one
neutral Texas Red dextran of 3000, 10,000, 40,000 or 70,000 daltons
(Invitrogen, Carlsbad, CA) diluted in ASW. Ten minutes after exposure to the
chamber, zygotes were imaged at the equatorial plane of each fertilization
envelope for both the Cascade Blue and Texas Red fluorophores, on a TCS SP2
AOBS confocal scanning microscope driven by proprietary software (Leica
Microsystems, Bannockburn, IL). Average fluorescence intensity was measured
over three random regions (30 pixel diameter) within the perivitelline space
or the surrounding medium using Metamorph software (Universal Imaging
Corporation, Downingtown, PA). Average Texas Red measurements per zygote were
normalized to Cascade Blue measurements per region per sample, and the
percentage of labeling in the perivitelline space versus the media was
calculated per region. Average percentages over three regions per zygote were
used to compare the population across 7-10 zygotes per treatment. Two-tailed
Student's t-tests were used to evaluate significance per treatment
compared with normal fertilization.
Localization and quantification of dityrosine formation
Peroxidase-mediated catalysis of dityrosine crosslinks within the
fertilization envelope was tracked with the fluorescent conjugates
tyramide-Alexa Fluor 488 and -Alexa Fluor 594 (Molecular Probes, Eugene, OR).
Stock solutions of tyramide-Alexa Fluor (made according to manufacturer's
instructions; the final concentrations are proprietary, thus we refer to the
concentrations as dilutions of this stock) were used in all experiments. A
final concentration of 1 mM 3-AT was used to inhibit ovoperoxidase as
necessary. Static imaging of the Alexa Fluor conjugates was accomplished on a
Zeiss LSM410 confocal laser-scanning microscope (Carl Zeiss Corporation,
Thornwood, NY) using RENAISSANCE software (Microcosm, Columbia, MD).
Quantification of tyramide-Alexa Fluor 488 incorporation within the
fertilization envelope was achieved by fertilizing eggs in the presence of a
1:200 dilution of the fluorochrome conjugate, as previously published
(Wong et al., 2004). Thirty
minutes after insemination, the cells were washed twice with ice-cold ASW, and
then stored on ice until visualization. Confocal microscope images containing
optical cross-sections of fertilization envelopes at the equator were analyzed
with Metamorph software (Universal Imaging Corporation, Downingtown, PA).
Fluorescence intensity was quantified as a ring from the cell surface to the
outside of the fertilization envelope: Total fluorescence intensity (I) and
total area (A) were measured in circular regions five pixels either outside
the fertilization envelope (FE) or along the cell plasma membrane (CELL).
Measurements for each species were normalized according to the following
formula:
 |
Time-lapse confocal microscopy was used to identify S. purpuratus
ovoperoxidase activity in vivo during fertilization envelope formation. For
these experiments, eggs were loaded into Kiehart chambers
(Kiehart, 1982) with a final
concentration of 1 µM FM1-43 (Molecular Probes), to label the cell
membrane, and a tyramide-Alexa Fluor 594 at a final dilution of 1:16,000. Eggs
within the chamber were inseminated with 4 µl of a 1:100 dilution of sperm
(1:10,000 final dilution). The field was then scanned every 10 seconds for the
membrane marker FM1-43 and tyramide-Alexa Fluor 594 accumulation using a TCS
SP2 AOBS confocal scanning microscope (Leica Microsystems, Bannockburn, IL).
The time when fertilization occurred (t=0) was determined by DIC
images, and a corresponding rise in FM1-43 fluorescence intensity at the cell
surface (Voronina and Wessel,
2004). Tyramide-Alexa Fluor 594 incorporation was then measured as
fluorescence intensity within the fertilization envelope per frame, as above.
Net fluorescence was calculated by subtracting background fluorescence values
at the image just prior to fertilization (t=-10 seconds). Time series
data sets per egg were normalized to the maximum fluorescence after 10 minutes
(600 seconds) of recording.
Labeling of ovoperoxidase substrates within the fertilization envelope
S. purpuratus eggs were equilibrated to 5 mM tyramine HCl and
various stock dilutions of either tyramide-Alexa Fluor 594 or tyramide-DSB
biotin (Invitrogen) dissolved in ASW before insemination. Thirty minutes after
insemination, zygotes were gently washed twice with ASW. Fertilization
envelopes were recovered from zygotes by passing them through a 64-µm nylon
mesh to separate the tyramine-softened fertilization envelopes (tyramine-SFEs)
from the cells. Cells were settled by gravity on ice, and the soft
fertilization envelopes (SFEs) in the supernatant were collected by
centrifugation at 10,000 g. SFEs were washed once with water
and then resuspended in 10 mM Tris, 50 mM EDTA, pH 8.0. This resuspension was
stored at -80°C until needed.
In vitro crosslinking of soft fertilization envelopes
S. purpuratus eggs were preincubated in 10 µM
diphenyleneiodonium (DPI; Sigma-Aldrich) and then fertilized as described
previously (Wong et al.,
2004). Non-crosslinked FEs from this method of inhibition
(DPI-SFEs) were separated and purified from zygotes with nylon mesh, as above,
and stored in a concentrated form in ASW at 4°C on ice until needed.
Samples treated with inhibitors such as 3-AT or DPI were incubated for 10
minutes at room temperature prior to the addition of hydrogen peroxide.
Various competitors, such as tyramine or tyramide analogs, were included in
specific experiments, as noted in figures. Crosslinking was achieved by
addition of exogenous hydrogen peroxide (10 nM to 10 µM final
concentration) diluted in ASW at room temperature. Following 20 minutes of
hydrogen peroxide exposure, each sample was washed twice in a 10-fold excess
of ASW. Treated SFEs were pelleted at 10,000 g for 5 minutes,
and stored dry at -80°C until needed.
Identification of ovoperoxidase protein targets within the fertilization envelope
Sixty micrograms of each SFEs sample was subjected to SDS-PAGE on pre-cast
4-20% polyacrylamide Tris-Glycine gels (Life-Therapeutics, Frenchs Forest, New
South Wales, Australia). Alexa Fluor 594 fluorescence was recorded using a
Typhoon fluorescent scanner run by proprietary software (Amersham Biosciences,
Piscataway, NJ). The gel was subsequently stained with Coomassie Blue to check
loading.
Alternatively, the separated proteins were transferred to nitrocellulose
for detection of DSB-biotin labeling (60 µg each SFE sample) or individual
components of the sea urchin extracellular matrix (2 µg each SFE sample).
Blots destined for DSB-biotin detection were blocked in 1% BSA in TBST [170 mM
NaCl, 50 mM Tris, 0.05% Tween20 (v/v), pH 8.0], then probed overnight with
Extravidin-AP (1:300,000 dilution; Sigma-Aldrich). Immunoblots were blocked in
Blotto [3% non-fat dry milk (w/v), 170 mM NaCl, 50 mM Tris, 0.05% Tween20
(v/v)], and then probed overnight with rabbit antisera against proteoliaisin
(Somers et al., 1989;
Somers and Shapiro, 1991),
SFE1 (Wessel et al., 2000a),
SFE9 (Wessel, 1995), separate
epitopes of rendezvin (Wong and Wessel,
2006b), ovoperoxidase
(LaFleur, Jr et al., 1998), or
fertilization envelope-incorporated vitelline layer (J.L.W. and G.M.W.,
unpublished). These immunoblots were washed twice with Blotto, then incubated
with alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich),
diluted 1:30,000 in Blotto, for 1 hour. All blots were washed extensively in
TBST, and then in alkaline phosphatase buffer (100 mM NaCl, 5 mM
MgCl2, 100 mM Tris, pH 9.5). Immunoreactivity of the secondary
antibody was detected by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue
tetrazolium (BCIP/NBT), as previously described
(McGadey, 1970).
|
) were
eluted directly into a QSTAR XL hybrid qTOF mass spectrometer (Applied
Biosystems, Foster City, CA and Sciex, Concord, Ontario, Canada) via
electrospray ionization (Josic et al.,
2006; Wilm et al.,
1996). LC-MS spectra were analyzed using MASCOT software
(Perkins et al., 1999). |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Furthermore, although barriers examined in mammals show increased permeability
to some serum proteins compared with exogenous proteins or dextrans of similar
molecular mass (Ambati et al.,
2000; Parr and Parr,
1986), the osmolarity of the sea urchin media caused similar
globular proteins to adhere to the fertilization envelope (data not shown),
possibly altering its filtration dynamics. We therefore used various dextrans.
The diffusion of Texas Red-labeled dextrans of various molecular mass across
the fertilization envelope and into the perivitelline space was compared with
the passage of 3000 dalton Cascade-Blue dextran. Both Strongylocentrotus
purpuratus and Lytechinus variegatus fertilization envelopes
possess a diffusion cut-off of 50% with 30,000- to 50,000-dalton dextrans
(Fig. 1), a shared threshold
that is consistent with their orthologous constituents
(Wong and Wessel, 2004). This
molecular permeability of the acellular fertilization envelope matrix is
similar to limits determined for the mammalian sclera
(Ambati et al., 2000) and the
cell-mediated endometrial decidual zone at the site of implantation
(Parr and Parr, 1986).
We next tested how perturbations to the formation of the fertilization
envelope affect filtration dynamics. A fertilization envelope assembled in
calcium-free seawater is morphologically similar to the original, highly
permeable vitelline layer (Carroll et al.,
1986; Cheng et al.,
1991). The source of this open architecture is likely to be the
decreased cohesion of the structural proteins in the absence of calcium
(Hall, 1978), and is thus a
control for the loss of restricted permeability. In addition, the absence of
calcium represses transglutaminase-dependent crosslinking
(Ha and Iuchi, 1998;
Lorand and Graham, 2003;
Zanetti et al., 2004),
allowing us to avoid the typical competitive substrate inhibitors for
transglutaminase, such as cadaverine, putresceine and glycine ethyl esters
(Battaglia and Shapiro, 1988),
which all have potential steric effects that could affect filtration dynamics.
By contrast, direct inhibitors of ovoperoxidase, such as 3-aminotriazole
(3-AT) (Foerder and Shapiro,
1977), enabled us to test the effects of dityrosine crosslinks on
permeability. In the absence of calcium
(Fig. 1C) or ovoperoxidase
activity (Fig. 1D), the
corresponding fertilization envelopes are no longer restrictive to the higher
molecular mass dextrans. Furthermore, the specificity of 3-AT for
ovoperoxidase activity (Foerder and
Shapiro, 1977) suggests that dityrosine crosslinks restrict 30-60%
diffusion of the high molecular mass dextans across the fertilization
envelope. Despite the facilitated delivery of 70,000-120,000 dalton
biomolecules upon inhibition of ovoperoxidase, the physical presence and
charge of the fertilization envelope still limit the accessibility of larger
biomolecules (e.g. DNA, RNA or proteins, such as immunoglobulins; J.L.W. and
G.M.W., unpublished) to the embryo; only complete removal of the fertilization
envelope will ensure maximal embryo exposure.
|
;
Bobrow et al., 1991;
Hunyady et al., 1996) to
visualize the in vivo targets of the endogenous sea urchin ovoperoxidase.
Inclusion of tyramide-Alexa Fluor in media during fertilization of either
S. purpuratus or L. variegatus eggs resulted in the
selective accumulation of fluorochrome within the fertilization envelope, but
not at the egg surface (Fig.
2C). Labeling with tyramide-Alexa Fluor is abolished in the
presence 3-AT (Fig. 2C,D)
(Showman and Foerder, 1979),
consistent with the localization of ovoperoxidase to the fertilization
envelope following cortical granule exocytosis
(Mozingo et al., 1994;
Somers et al., 1989).
Time-lapse imaging of fertilization in the presence of
tyramide-fluorochrome conjugates revealed a biphasic activity curve for S.
purpuratus ovoperoxidase (Fig.
3). Enzyme crosslinking is linear for about 275 seconds following
fertilization, and then plateaus over the remaining 225 seconds; the time to
50% completion is approximately 140 seconds
(Fig. 3B). This biphasic
profile is a direct effect of hydrogen peroxide synthesis by Udx1
(Wong et al., 2004), such that
the linear phase overlaps the most abundant production of hydrogen peroxide
and the lagging phase corresponds to the depression of Udx1 activity
(Fig. 3B). Thus, the
rate-limiting step of ovoperoxidase crosslinking of fertilization envelope
proteins is the availability of its primary substrate. Although the majority
of the detectable nine million molecules of hydrogen peroxide synthesized by a
single embryo (Wong et al.,
2004) is used to establish dityrosine crosslinks, not all of these
molecules are consumed by ovoperoxidase; the remainder may be used in cell
signaling (Wong and Wessel,
2005), as a catalytic `sterilizing' agent
(Klebanoff et al., 1979), or
may simply decay during its diffusion away from the embryo.
Identification of endogenous ovoperoxidase targets
Isolation and identification of peroxidase substrates requires that the
targets be soluble, yet a common outcome of crosslinking is matrix
insolubility - as exemplified by the gel mobility of the fertilization
envelope constituent SFE9 (Wessel,
1995). To counter this, we used competitive substrates that formed
free tyrosyl radicals (Gulyas and Schmell,
1980; Jacob et al.,
1996) to inhibit endogenous, inter-protein dityrosine
crosslinking. We first tested the effects of free L-tyrosine, but, as noted
previously (Hall, 1978), its
low solubility in media proved problematic. In the presence of the
tyramide-fluorochrome precursor tyramine, however, we observed a specific and
dose-dependent increase in SFE9 gel mobility compared with the soluble loading
control YP30 (Wessel et al.,
2000b). The performance of 5 mM tyramine is equal to the
inhibition of ovoperoxidase activity achieved with 1 mM 3-AT
(Showman and Foerder, 1979)
(see Fig. S1 in the supplementary material); indeed, we were able to separate
these tyramine-treated soft fertilization envelopes (tyramine-SFEs) from the
zygotes or embryos with gentle mechanical shearing (see Fig. S2A in the
supplementary material). Furthermore, the presence of excess tyramine does not
affect the retention of tyramide-Alexa Fluor in the fertilization envelope
(see Fig. S2 in the supplementary material).
|
) or carboxy-domains (RDZ
anti-
) of rendezvin (Wong and
Wessel, 2006b) shows that only the carboxyl terminus of rendezvin
(RDZ120) co-migrates with this fluorescent band. To determine if
this is true in situ, we used an unbiased crosslinking assay probing whole
cell homogenates of zygotes fertilized in the presence of either the direct
ovoperoxidase inhibitor 3-AT or the dual oxidase inhibitor diphenyleneiodonium
(DPI), which indirectly inhibits ovoperoxidase crosslinking by blocking
substrate availability (Showman and
Foerder, 1979; Wong et al.,
2004). Under these conditions, the gel mobility of the vitelline
layer-associated protein RDZ120 is impaired when ovoperoxidase
activity is present, thus supporting its role as a major substrate of this
peroxidase (Wong and Wessel,
2006b). RDZ60 and RDZ90, however, are not
substrates of ovoperoxidase, revealing distinct selectivity in the target
recognition of this enzyme.
We also developed a semi-in vivo crosslinking assay to assess the mechanism
of fertilization envelope modification. We used the observation that
fertilization envelope constituents remain in complex following isolation in
the absence of ovoperoxidase activity
(Wong and Wessel, 2006b).
Pliable fertilization envelopes were isolated in the presence of DPI so that
ovoperoxidase was not affected, and then exposed to exogenous hydrogen
peroxide for 20 minutes (Fig.
5A) to simulate the timing of Udx1 activity in vivo
(Wong et al., 2004). This
semi-in vivo system significantly enhanced the sensitivity with which the
target population could be identified without compromising ovoperoxidase
function, as revealed by the select labeling of the slower-migrating
components proteoliaisin, SFE1 and SFE9
(Fig. 5B,C). The visualization
of LDLrA-containing proteins (Wong and
Wessel, 2004) as ovoperoxidase targets is consistent with previous
observations (Wessel, 1995)
and supports our mass spectrometry data obtained for a high molecular mass
complex (data not shown). Again, neither RDZ60 nor RDZ90
are crosslinked under the physiological concentrations of hydrogen peroxide
used (Fig. 5D), further
reinforcing the molecular selectivity of ovoperoxidase activity.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
production of active thyroid hormone by thyrocytes
(Dunn and Dunn, 2001;
Yip, 1966) both rely on
peroxidase-mediated crosslinking events for normal function. Pathologically,
pulmonary fibrosis resulting from excess dityrosine crosslinked lesions
between collagen and elastin have been linked to transforming growth factor
beta 1 (TGFβ1)-stimulated peroxidase release from lung fibroblasts during
local inflammation (Larios et al.,
2001), whereas atherosclerosis is associated with excess spurious
tyrosyl conjugates generated by myeloperoxidase released from neutrophils at a
site of inflammation (Heinecke,
1999; Heinecke et al.,
1993a; Heinecke et al.,
1993b; Jacob et al.,
1996). The reactive free radicals formed by this enzyme family are
also correlated with molecular damage linked to aging
(Bergendi et al., 1999;
Fridovich, 1998;
Levine and Stadtman, 2001;
Linton et al., 2001).
|
;
Li et al., 1996). Although the
formation of di- and tri-tyrosine conjugates have been identified by methods
such as endogenous ultraviolet absorbance, chromatography and mass
spectrometry (Heinecke, 1999;
Heinecke et al., 1993a;
Heinecke et al., 1993b;
Jacob et al., 1996;
Larios et al., 2001), the
identity of the parent extracellular matrix targets remains a mystery. In
fact, the free radical intermediate
(Davies and Delsignore, 1987;
Davies et al., 1987) suggests
that the mechanism of crosslinking simply depends on the abundance and
accessibility of tyrosine residues, rather than on peroxidase target
specificity. Within the sea urchin fertilization envelope, however, the free
radical chemistry is clearly selective. In contrast to the apparent
promiscuity of ovoperoxidase to exogenous substrates such as bovine serum
albumin (Klebanoff et al.,
1979), we find here that only a subset of the endogenous
fertilization envelope proteins are in vivo substrates. Indeed, sibling
proteins, resulting from differentially spliced rendezvin mRNAs
(Wong and Wessel, 2006b), are
differentially crosslinked within the fertilization envelope. This preference
for proteins - versus halides (Morrison
and Schonbaum, 1976), free amino acids
(Gross, 1959) or lipids
(Levine and Stadtman, 2001) -
is likely a consequence of tyrosyl abundance and availability within the
fertilization envelope, a factor further enhanced by the tethering of
ovoperoxidase to this matrix by proteoliaisin
(Somers et al., 1989). Thus,
substrate specificity and the quantity of total residues affected
(one-crosslink per 50-100 kDa total protein)
(Foerder and Shapiro, 1977;
Hall, 1978) are likely to be
essential for maintaining fertilization envelope permeability until
hatching.
|
). (2)
Environmental alkalination auto-activates these cortical granule-derived
proenzymes over different time frames
(Deits and Shapiro, 1985;
Haley and Wessel, 2004b).
CGSP1 activates first, and proceeds to (3) cleave the microvillar protein p160
(Haley and Wessel, 1999;
Haley and Wessel, 2004a),
freeing the vitelline layer so that it separates from the plasma membrane by
the hydrating force of cortical granule-derived mucopolysaccharides released
between the zygotic hyaline layer and the fertilization envelope
(Harvey, 1909;
Larabell and Chandler, 1991).
(4) Egg surface transglutaminase at the microvillar caps crosslinks local
glutamines and lysines (Battaglia and
Shapiro, 1988). This activity requires cortical granule
exocytosis, suggesting that CGSP1 proteolysis may activate zymogenic
transglutaminase - similar to the activity of thrombin on factor XIIIa during
blood clotting (Verderio et al.,
2004). Transamidation tapers
(Battaglia and Shapiro, 1988)
as (5) synthesis of hydrogen peroxide by Udx1 peaks
(Wong et al., 2004) and
ovoperoxidase overcomes its pH-dependent conformational hysteresis
(Deits and Shapiro, 1985;
Deits and Shapiro, 1986).
These coordinated events provide optimal rates of dityrosine crosslinking,
resulting in covalent bonding of 13-15% of all available tyrosyl residues
within the fertilization envelope (Foerder
and Shapiro, 1977; Hall,
1978). (6) Covalent crosslinking activity mostly affects the
ovoperoxidase-tether proteoliaisin and its direct binding partner, the
vitelline layer-associated isoform of rendezvin (RDZ120), which
preferentially associates with the other low-density lipoprotein type A
(LDLrA) repeat-containing proteins SFE1 and SFE9
(Mozingo et al., 1994;
Somers and Shapiro, 1991;
Weidman et al., 1985;
Wong and Wessel, 2006b). This
local clustering of target proteins within discrete zones within the
fertilization envelope (Kay and Shapiro,
1987; Mozingo and Chandler,
1991; Mozingo et al.,
1994; Somers and Shapiro,
1989) is likely to be a major contributor to the selectivity of
ovoperoxidase, although CGSP1 and transglutaminase may have also modified the
matrix sufficiently to restrict access of ovoperoxidase to other proteins,
including the cortical granule rendezvin components RDZ60 and
RDZ90. After 2 minutes of peak activity, repression of Udx1
(Wong et al., 2004) and
prolonged CGSP1 activity (Haley and
Wessel, 2004b) together inhibit further crosslinking. Thus, the
tight regulation of ovoperoxidase activity and restricted localization
together transform the uniform matrix assembly into a barrier that encloses
the embryo in a microenvironment critical for development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/3/431/DC1
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ACKNOWLEDGMENTS |
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