From the Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205
Received for publication, February 21, 2001, and in revised form, April 4, 2001
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ABSTRACT |
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Fertilin ADAM1 (for A disintegrin and
A metalloprotease) proteins comprise a recently
identified and rapidly growing molecular family of membrane proteins.
To date, ~30 ADAMs have been identified in a variety of animal
species, including several mammals, Xenopus laevis,
Drosophila melanogaster, and Caenorhabditis
elegans (1, 2). The conserved domain structure of ADAMs (see Fig.
1A) and functional analyses of these proteins indicate that
members of this family have functions as proteases and/or as cell
adhesion molecules. In the ADAMs that have been described to function
as cell adhesion molecules, the adhesive activity generally appears to
be attributable to the disintegrin-like domain, a domain with homology
to integrin ligand-like snake venom polypeptides. These venom
polypeptides, known as disintegrins, contain an RGD tripeptide, presented at the end of an extended loop structure called the "disintegrin loop" (3). Although ADAMs share significant sequence homology in their disintegrin-like domains to snake venom disintegrins, they do not have RGD sequences in their putative disintegrin loops (with the exception of human ADAM15). Various studies have shown that
the adhesion-mediating sequence of several ADAMs appears to be a short
sequence within the putative disintegrin loop, such as the
ECD-containing sequences in fertilin Among the cell adhesion events that are mediated by ADAMs are the
interactions between mammalian gamete plasma membranes during fertilization. On mouse sperm, there are at least three ADAMs that
appear to participate in sperm-egg binding: fertilin The primary purpose of this study was to test the hypothesis that the
fertilin In light of this finding and other studies demonstrating that
function-blocking antibodies against the Production of Recombinant Fertilin Proteins--
The complete
extracellular portion of fertilin Synthetic Peptides--
Peptides corresponding to different
parts of the disintegrin domain of fertilin Bacterial Alkaline Phosphatase-presented Peptides--
Bacterial
alkaline phosphatase (BAP)-presented peptides were generated as
described previously (4). Oligonucleotide sequences that were used are
shown in Table I. In brief, complementary oligonucleotides were annealed to each other and ligated into the
XbaI and BglII restriction sites in the multiple
cloning site of the plasmid pHNAP, which includes a 6-histidine tag.
Fusion proteins were expressed and purified on a nickel column as
described previously (4).
Egg Collection, Zona Pellucida Removal, and IVF--
Metaphase
II-arrested eggs were obtained from superovulated 6-9-week-old CF-1
mice (Harlan, Indianapolis, IN); egg collection and cumulus cell
removal were performed as described previously (4). All gamete cultures
were performed in Whitten's medium (20) containing 22 mM
NaHCO3 and 15 mg/ml bovine serum albumin (Albumax I; Life
Technologies, Inc.) at 37 °C in 5% CO2 in air. For most
experiments, the zona pellucida (ZPs) were removed from eggs via
solubilization with a very brief incubation ( Luminometric Immunoassay to Quantify the Binding of Recombinant
Fertilin Proteins to Eggs--
To determine the effects of synthetic
peptides or BAP-presented peptides on the binding of recombinant
fertilin Assessment of the Binding of Bead-immobilized Recombinant
Fertilin Proteins to Eggs--
Fluorescent beads (0.2-µm
yellow-green sulfate-derived latex FluoSpheres; Molecular Probes,
Eugene, Oregon) were coated with recombinant fertilin Effects of Different Recombinant Forms of Fertilin Effects of Synthetic Peptides Corresponding to Different Portions
of the Fertilin
These three experimental synthetic peptides and their accompanying
control peptides were tested for their ability to inhibit sperm-egg
binding during IVF (Fig. 4). Of these six
synthetic peptides, Peptide a-3 was the only one that inhibited
sperm-egg binding (reducing binding to ~50% of levels observed in
the no peptide control and in peptide a-3s; p < 0.05).
Effects of Synthetic and BAP-presented Peptides Corresponding to
Different Portions of the Fertilin
The effects of the synthetic peptides on the binding of recombinant
fertilin
We also tested the a-3 sequence (DLEECDCG) and the a-1 sequence
(AEDVCDLP) in different forms, as BAP-presented peptides. The rationale
for this was previous work in our lab in which we found that a
BAP-presented peptide with the sequence corresponding to the fertilin
The effects of the BAP-presented peptides on recombinant fertilin
We also used a different protein binding assay to confirm the effect of
BAP-a-3 on recombinant fertilin Effects of Synthetic and BAP-presented a-3 Peptides on the Binding
of Recombinant Fertilin
Peptide a-3, as a synthetic peptide and as a BAP-presented peptide,
perturbed the interaction of recombinant fertilin
We also assessed the effect of BAP-a-3 on bead-immobilized recombinant
fertilin Effects of Anti-
We also examined the effects of GoH3 and KMC.8 on the binding of
recombinant fertilin In this study, we have demonstrated that the disintegrin-like
domain of mouse fertilin This a-3 region in fertilin The DLEECDCG a-3 sequence inhibits the binding of not only recombinant
fertilin We also present data indicating that two different regions of fertilin
These data suggest that there may be similarities between fertilin
With regard to egg binding partners for fertilin (also known as ADAM1) is a member of
the ADAM (A disintegrin and A
metalloprotease domain) family of proteins. In this study,
we examine the mechanism of mouse fertilin
's in adhesion of sperm
to the egg plasma membrane during fertilization. We find that
recombinant forms of fertilin
corresponding to either the
disintegrin-like domain or the cysteine-rich domain and the EGF-like
repeat can perturb sperm-egg binding, suggesting that both of these
domains can participate in fertilin
-mediated adhesion events. In
further examination of the fertilin
disintegrin-like domain, we
find that a subdomain of disintegrin-like domain with the sequence
DLEECDCG outside the putative disintegrin loop but with homology to the
fertilin
disintegrin loop can inhibit the binding of both sperm and
recombinant fertilin
to eggs, suggesting that this is an
adhesion-mediating motif of the fertilin
disintegrin-like domain.
This sequence also inhibits the binding of recombinant fertilin
to
eggs and thus is the first peptide sequence found to block two
different sperm ligands. Finally, a monoclonal antibody to the
tetraspanin protein CD9, KMC.8, inhibited the binding of recombinant
fertilin
to eggs in one type of binding assay, suggesting that,
under certain conditions, fertilin
may interact with a KMC.8-sensitive binding site on the egg plasma membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(4, 5), ADAM9 (6), and ADAM23
(7), or the QCD-containing sequence in cyritestin (8).
(ADAM1), fertilin
(ADAM2), and cyritestin (ADAM3) (reviewed in Ref. 9). As
noted above, fertilin
and cyritestin, as well as several other
ADAMs, appear to mediate cell adhesion through short peptide sequences
within the putative disintegrin loop of the disintegrin-like domain (4,
5, 8). With regard to fertilin
, studies of the putative disintegrin
loop have been inconclusive, with a synthetic peptide corresponding to
the fertilin
disintegrin loop (AEDVCDLP) having a modest inhibitory
effect on sperm-egg interactions but with the scrambled control peptide
(PDCEVADL) also having some effect (8). Nevertheless, there are data
that imply that the fertilin
disintegrin-like domain participates in cell adhesion but also that the cysteine-rich domain can mediate adhesion as well. A recombinant form of fertilin
corresponding to
the complete extracellular domain (i.e. the disintegrin-like domain, the cysteine-rich domain, and the EGF-like
repeat) inhibits sperm-egg binding more
effectively than does a shorter form with a truncated disintegrin-like
domain (10), suggesting that the presence of disintegrin-like domain in
recombinant fertilin
enhances the ability of the recombinant
fertilin
protein to inhibit sperm-egg binding. However, this
truncated recombinant fertilin
, containing only the last C-terminal
20 amino acids of the disintegrin-like domain, along with the
cysteine-rich domain, and the EGF-like repeat (designed with respect to
the original N-terminal sequence data (11)), still binds to mouse eggs
and inhibits sperm-egg binding (12). In agreement with this, an antibody against a similar construct of recombinant rabbit fertilin
, with a truncated disintegrin-like domain, inhibits rabbit in vitro fertilization (IVF) (13). These data suggest that domains in
fertilin
other than the disintegrin-like domain, namely the cysteine-rich domain and/or the EGF-like repeat, participate in sperm-egg adhesion. Interestingly, this is similar to another member of
the ADAM family, ADAM12, which appears to be able to utilize multiple
domains to mediate cell adhesion, based on the observation that
recombinant forms of the ADAM12 cysteine-rich domain (14) and the
ADAM12 disintegrin-like domain (15) can support cell adhesion in
vitro.
disintegrin-like domain contains amino acids that interact
with egg-binding sites. Our results with recombinant fertilin
disintegrin-like domain (hereafter referred to as recombinant fertilin
D) suggest that the fertilin
disintegrin-like domain participates in fertilin
-mediated sperm-egg binding. An examination of candidate subdomains of the fertilin
disintegrin-like domain to
characterize what region(s) of the disintegrin-like domain had adhesive
activity identified a short adhesion-mediating sequence that has
homology to the fertilin
disintegrin loop but is in a location
distinct from the putative disintegrin loop region of fertilin
.
6 integrin
subunit or against the tetraspanin protein CD9 inhibit sperm and
fertilin
binding to eggs (16-18), we also investigated the
possibility that function-blocking antibodies against either of these
proteins on eggs (GoH3 against
6; KMC.8 against CD9)
would inhibit the binding of the recombinant fertilin
D to eggs. We
find that GoH3 has no effect but that KMC.8 reduces recombinant
fertilin
D binding detected by one type of assay. This observation
suggests that the fertilin
disintegrin-like domain could interact
with a KMC.8-sensitive site on the egg membrane but also indicates that
the mode of presentation of recombinant fertilin
affects the
sensitivity of fertilin
to inhibition by KMC.8.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(recombinant fertilin
DCE)
included amino acids 406-708 (counting the start Met as amino acid 1)
and was encoded by nucleotides 1216-2124 (counting the start ATG codon
of full-length mouse fertilin
, assembled from the two partial
cDNA sequences (accession numbers AF167406 and U22056), as
nucleotides 1-3). This cDNA region was prepared by polymerase
chain reaction (PCR) amplification using a 5' primer corresponding to
nucleotides 1216-1232 engineered with a XbaI restriction
site (GGTCTAGAGCTGCCAATTGTGGGAA) and a 3' primer corresponding to
nucleotides 2109-2124 engineered with a stop codon and a
SalI restriction site (TTTTGTCGACTTATTTCAGGTTTACCTCT). The
region encoding the extracellular portion of fertilin
lacking the
disintegrin domain (recombinant fertilin
CE, amino acids 493-708,
encoded by nucleotides 1477-2124) was prepared by PCR amplification
using a 5' primer corresponding to nucleotides 1477-1493 engineered
with a XbaI restriction site (GGTCTAGACAGTGTGATAGGATTTA) and
a 3' primer corresponding to nucleotides 2109-2124 engineered with a
stop codon and a SalI restriction site
(TTTTGTCGACTTATTTCAGGTTTACCTCT). The region encoding the fertilin
disintegrin-like domain (recombinant fertilin
D, amino acids
406-493, encoded by nucleotides 1216-1478) was prepared by PCR
amplification using 5' primer corresponding to nucleotides 1216-1232
engineered with a XbaI restriction site (GGTCTAGAGCTGCCAATTGTGGGAA) and a 3' primer corresponding to
nucleotides 1463-1479 engineered with a stop codon and a
SalI restriction site (TTTTGTCGACTTACTGTGTGCCATCCTGCA). PCR
amplifications using mouse fertilin
cDNA (pVAC-
.2) as
template were performed in a PEC 2400 thermocycler (PerkinElmer Life
Sciences) with Pfu I polymerase (Promega, Madison, WI). The
PCR products were then digested with XbaI and
SalI and cloned into pMAL-p.2 (New England Biolabs, Beverly,
MA) as described previously (10, 12, 19). The resulting plasmids were
sequenced to verify that the DNA sequence encoded the correct amino
acid sequence. All recombinant fertilin-maltose-binding protein (MBP)
fusion proteins were expressed in DH5
Escherichia coli
cells with induction by
isopropyl-1-thio-
-D-galactopyranoside, then purified on
an amylose affinity column, and renatured by reduction and oxidation as
previous described (10, 12, 19).
were synthesized by
Alpha Diagnostic International (San Antonio, TX) and purified by high
pressure liquid chromatography to
95% purity. The N termini were
acetylated, and the C termini were amidated. The sequences were chosen
based on sequence alignment to the RGD-containing disintegrin loops of
snake venom disintegrins (peptide a-1; AEDVCDLP, amino acids 465-472
of full-length mouse fertilin
), because of conservation between
fertilin
and cyritestin (peptide a-2; DLPEYCDG, amino acids
470-477) or because of homology to the disintegrin loop of fertilin
(peptide a-3; DLEECDCG, amino acids 416-423). The scrambled
peptide controls contained the same eight amino acids in rearranged
order (a-1s, EPCVDALD; a-2s, EGDCPLDY; a-3s; CELDGDCE), respectively.
Peptides were resuspended in degassed, sterile water to a stock
concentration of 5 mM and then lyophilized in aliquots such
that a 25-µl resuspension in Whitten's culture medium (20) would
yield a final peptide concentration of 1 mM.
Oligonucleotides used to generate BAP-presented peptides
15 s) in acidic
medium-compatible buffer (4). For experiments using the monoclonal
antibody GoH3 (anti-
6 integrin subunit), the ZP were
removed by incubating the eggs in medium containing 10 µg/ml chymotrypsin (Sigma) for 5 min, allowing the ZP to swell, and then
shearing off the ZP with a thin bore pipette. This treatment is used
because GoH3 does not bind to eggs from which the ZP has been removed
by acid solubilization (19). Following ZP removal, the eggs were
allowed to recover for 60 min in Whitten's medium. Sperm were
collected from the caudae epididymides of (C57BL/6J × SJL/J)F1
male mice (8-10 weeks old; Jackson Laboratories, Bar Harbor, ME) and
capacitated in vitro for 2.5-3 h. To examine the effects of
recombinant fertilin
proteins on sperm-egg interactions, ZP-free
eggs (20-25 eggs/5-6.5-µl drop) were incubated for 60 min prior to
insemination in Whitten's medium containing 0.5 mg/ml MBP, recombinant
fertilin
DCE, recombinant
D, or recombinant
CE. To examine the
effects of synthetic peptides on sperm-egg interactions, ZP-free eggs
(20-25 eggs/5-6.5-µl drop) were incubated for 60 min prior to
insemination in Whitten's medium containing 1.0 mM of
synthetic peptide a-1, a-1s, a-2, a-2s, a-3, or a-3s for 60 min. After
incubation of eggs in either the peptide or the recombinant fertilin
protein, 5-6.5 µl of a suspension of capacitated sperm were
added so that the final sperm concentration was 500,000 sperm/ml
(adapted from Ref. 21). The final concentration of the peptides during
insemination was 0.5 mM, and the final concentration of the
recombinant proteins during insemination was 0.25 mg/ml. After a 15-min
insemination, the eggs were washed three times to remove loosely
attached sperm (adapted from Ref. 21). The eggs were then fixed in
3.7-4.0% paraformaldehyde for 30 min, permeabilized with 0.1% Triton
X-100 for 5 min, and mounted in VectaShield mounting medium (Vector
Labs, Burlingame, CA) containing 1.5 µg/ml
4',6-diamidino-2-phenylindole (Sigma) to stain the sperm DNA.
The average number of sperm bound per egg was determined. The values
from each experiment were normalized with respect to the appropriate
control (i.e. the MBP control or the no peptide control was
defined as 100%). The data from multiple IVF experiments were analyzed
by analysis of variance with Fisher's protected least significant
difference post-hoc testing using StatView version 5.0 (SAS
Institute, Cary, NC). A value of p < 0.05 was
considered significant.
or fertilin
, ZP-free eggs were first incubated in the
indicated synthetic peptide (at 0.5 mM) or BAP-presented
peptide at the indicated concentration (20, 40, or 80 µM)
in Whitten's medium containing 15 mg/ml bovine serum albumin for 60 min and then in Whitten's medium containing both 0.5 mg/ml of the
indicated form of recombinant fertilin
or fertilin
and the
indicated BAP-presented peptide for an additional 60 min. During these
incubations, the culture medium was supplemented so that the final
concentrations of CaCl2 and MgSO4 were 2.4 and
1.2 mM, respectively. The binding of MBP recombinant
fertilin fusion proteins to ZP-free eggs was quantified as described
previously (4), using an anti-MBP polyclonal rabbit antiserum (diluted
1:750; New England Biolabs), followed by an alkaline
phosphatase-conjugated secondary antibody (alkaline
phosphatase-conjugated goat anti-rabbit IgG at 0.12 µg/ml; Jackson
Immunoresearch, West Grove, PA). Egg-associated alkaline phosphatase
activity was detected using the photon-emitting AP substrate disodium
3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan)-4-yl)phenyl
phosphate (Tropix, Bedford, MA). Photon emission was measured in
raw light units/10 s in a Monolight 3010 luminometer (Analytical
Luminescence Laboratory, Sparks, MD). The data were normalized with
respect to the appropriate control (indicated in the figure legends).
Statistical analyses by analysis of variance with Fisher's protected
least significant difference post-hoc testing were performed
using StatView version 5.0 (SAS Institute).
D (see Fig.
1C) or recombinant fertilin
D (4) as follows. The beads
(5 µl of a 2% bead suspension) were incubated with 5 µl of 2 mg/ml
anti-MBP IgG (purified by chromatography on protein G beads (Life
Technologies, Inc.) from monoclonal clone MBP-17 ascites fluid (Sigma))
for 2 h. The beads were then washed once by resuspending them in
phosphate-buffered saline followed by centrifugation at 4500 × g for 20 min. Recombinant fertilin
D or
D was then
added to the beads (at a final concentration of 1 mg/ml in a total
volume of 15 µl), and the beads were incubated overnight at 4 °C.
The beads were then incubated with rabbit IgG (final concentration, 1 mg/ml) for 1 h at room temperature. The beads were washed twice
with phosphate-buffered saline and resuspended to a concentration of a
0.2% bead suspension in Whitten's medium compatible buffer (109.5 mM NaCl, 4.7 mM KCl, 1.2 mM
KH2PO4, 7 mM NaHCO3, 15 mM HEPES; WHITCO). The 0.2% bead suspension was sonicated
three times for 30 s immediately prior to use and then diluted to
0.02% in Whitten's medium containing 15 mg/ml bovine serum albumin
and 100 µM of the indicated BAP-presented peptide. This
0.02% bead suspension was used to make 20-µl culture drops, to which
ZP-free eggs were added, following a 60-min incubation in medium
containing the indicated BAP-presented peptide at 100 µM.
The eggs were co-cultured with beads for 60 min at 37 °C in 5%
CO2. The eggs were washed four times through 100-µl drops
of Whitten's medium to remove unbound beads and were then mounted on a
microscope slide in Whitten's medium and viewed on a Nikon Eclipse
fluorescent microscope. The digital images were captured with a
Princeton 5-mHz cooled interlined CCD camera (Princeton Instruments
Inc., Trenton, NJ) using IP Lab (Scananalytics, Fairfax, VA) and
Photoshop (Adobe Systems Incorporated, San Jose, CA) software. The bead
binding levels were assessed qualitatively, estimated as compared with
binding levels in control groups, by examining eggs from the entire
experimental series (three experiments/series, with 10-20
eggs/group/experiment examined). The level of recombinant fertilin
D-coated bead binding to control eggs (see Fig. 7) appeared to be
higher than the level of binding of recombinant fertilin
D-coated
beads (see Fig. 6); this is in agreement with results using the
quantitative luminometric
assay.2
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
on Sperm-Egg
Binding--
We have previously demonstrated that a form of
recombinant fertilin
comprising amino acids 409-708 of mouse
fertilin
, including the disintegrin-like, cysteine-rich, and
EGF-like repeat domains, inhibited sperm-egg interactions (10). In that
study, we also tested a form of mouse fertilin
that corresponded to the originally reported N-terminal sequence of guinea pig fertilin
(11) comprising a truncated disintegrin-like domain (the C-terminal most 20 amino acids), the cysteine-rich domain, and the EGF-like repeat
(amino acids 474-708). This recombinant protein also perturbed sperm-egg binding, although not as effectively as the longer form encompassing amino acids 409-708 (10). To follow up on this observation, we used a recently developed, improved assay for sperm-egg
binding (21) and generated three forms of recombinant fertilin
to
examine more closely the role of the disintegrin-like domain in
fertilin
-mediated sperm-egg adhesion. Our updated form of
recombinant fertilin
DCE corresponded to amino acids 406-708, based
on the corrected N-terminal sequence of guinea pig and the N-terminal
sequence of bovine fertilin
(22, 23) (please note that this differs
slightly from our first version of recombinant fertilin
DCE (amino
acids 409-708) (10)). Our updated form of recombinant fertilin
CE
corresponded to the cysteine-rich domain and the EGF-like repeat of
mouse fertilin
(amino acids 493-708) (Fig.
1C); please note that the form
of
CE used in these experiments does not include any portion of the
disintegrin-like domain and thus differs from the form we used in
previous experiments (10, 12), which contained a truncated
disintegrin-like domain based on the original N-terminal sequence data
for guinea pig fertilin
(11). Recombinant fertilin
D
corresponded to the 88 amino acids (amino acids 406-493) of
disintegrin-like domain of mouse fertilin
(Fig. 1C). In
IVF experiments using these proteins, we observed that sperm-egg
binding was reduced in the presence of the three recombinant fertilin
forms (
DCE,
D, and
CE) as compared with sperm-egg binding
in the presence of the control protein, MBP (Fig.
2). (We have previously demonstrated that
sperm-egg binding and fusion are not affected by the presence of MBP in
the IVF culture medium (19).) In these studies, the levels of sperm-egg
binding in the presence of recombinant fertilins
DCE,
D, and
CE were not statistically significantly different from each
other.
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Fig. 1.
Schematic diagrams of domain structures of
fertilin and the recombinant fertilin
forms. A and B, domain
structures of full-length and mature fertilin
, based on the deduced
amino acid sequence from the cDNA clones (accession numbers U22056
and AF167406). The proteolytic cleavage site and the domain structure
of mature fertilin
(B) are predicted based on N-terminal
amino acid sequence data on guinea pig and bovine fertilin
(22,
23). C, domain structures of the MBP fusion proteins
corresponding to different regions of mature fertilin
.
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Fig. 2.
Effects of recombinant fertilin
proteins on sperm-egg binding during IVF.
ZP-free eggs were first incubated in medium containing 0.5 mg/ml of the
indicated protein (10.9 µM of MBP, 6.3 µM
of
DCE, 9.0 µM of
D, and 7.1 µM of
CE) for 60 min. The eggs were inseminated with 500,000 sperm/ml for
15 min (final protein concentration during the insemination was 0.25 mg/ml). The data presented are normalized with respect to the MBP
negative control (defined as 100%; in these experiments, the average
number of sperm bound per MBP-treated egg was 14.2 ± 3.03). This
graph represents data from five experiments, in which 20-30 eggs were
examined per group per experiment. The error bars represent
the S.E. The asterisks indicate p < 0.0.5 as compared with the MBP control. (The levels of sperm-egg binding in
DCE,
D, and
CE were not statistically significantly different
from each other.)
Disintegrin Domain on Sperm-Egg
Binding--
Because recombinant fertilin
D had an inhibitory
effect on sperm-egg binding (Fig. 2), we became interested in the
possibility that a specific subdomain(s) of the fertilin
disintegrin-like domain could mediate the interaction of sperm with the
egg plasma membrane. To investigate this possibility, three amino acid
sequences (Fig. 3) were tested as
synthetic peptides for their abilities to perturb sperm-egg binding
during IVF. Peptide a-1 (AEDVCDLP, amino acids 465-472) was identical
to the fertilin
peptide tested by Yuan et al. (8) and
corresponded to the putative disintegrin loop, lining up with the
RGD-containing region of snake venom disintegrins. Peptide a-2
(DLPEYCDG, amino acids 470-477) corresponded to a stretch of eight
amino acids just C-terminal from the putative disintegrin loop; this
amino acid sequence was chosen because it was well conserved in all
fertilin
homologs, in all cyritestin homologs, and in fertilin
homologs as well (see Table I in Ref. 9). In addition, the last four of
the eight amino acids of this highly conserved DLPEYCDG sequence were
present in the form of recombinant fertilin
with the truncated
disintegrin domain (amino acids 474-708) that inhibited sperm-egg
interactions (10, 12). Therefore, we hypothesized that this DLPEYCDG
sequence of fertilin
could be involved in sperm-egg interactions.
Finally, peptide a-3 (DLEECDCG, amino acids 416-423) was chosen
because it showed homology to the consensus sequence of the fertilin
disintegrin loop, X(D/E)ECD (where X is any
amino acid); the ECD sequence was demonstrated to be the key
adhesion-mediating motif, with the Asp residue being particularly
important (4, 5). For all of these peptides, we designed scrambled
controls designated a-1s (EPCVDALD), a-2s (EGDCPLDY), and a-3s
(CELDGDCE), respectively. For peptide a-1s, we chose a sequence
slightly different from the fertilin
disintegrin loop scrambled
peptide control used by Yuan et al., because their scrambled
control peptide showed a slight level of inhibition (8).
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Fig. 3.
Disintegrin-like domains of fertilin
homologs and design of peptide sequences.
A shows the alignments of the amino acid sequences of the
disintegrin-like domains of mouse (M), guinea pig
(GP), macaque (Mac), rabbit (Rab),
rat, bovine (Bov), orangutan (Oran), and baboon
(Bab) fertilin
. Shown below these is the consensus
(Con) sequence. The VCD tripeptide sequence (in bold
type) corresponds to the putative disintegrin loop. The EECD
sequence (in bold type and underlined) has
homology to the DECD in the fertilin
disintegrin loop. The LPEYC
adjacent to the VCD in the putative disintegrin loop
(underlined with a dotted line) is highly
conserved in all species orthologs of fertilin
(also shown in the
consensus sequence). B shows the amino acid sequences of
peptides a-1, a-2, and a-3 relative to where they align with respect to
the mouse fertilin
amino acid sequence. Peptide a-1 corresponds to
the putative disintegrin loop within disintegrin-like domains
(overlapping with the RGD-containing region of true disintegrins). The
numbers above the sequences refer to the
numbering of the mouse fertilin
amino acid sequence (assembled from
the two partial cDNA sequences; accession numbers AF167406 and
U22056), counting the methionine encoded by the start codon as amino
acid 1.
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Fig. 4.
Effects of synthetic peptides a-1, a-2, and
a-3 on sperm-egg binding during IVF. ZP-free eggs were incubated
in 1.0 mM of the indicated synthetic peptide (a-1, a-1s,
a-2, a-2s, a-3, or a-3s) for 60 min. The eggs were then inseminated in
the presence of synthetic peptide (at a final concentration of 500 µM) with 500,000 sperm/ml for 15 min. The eggs were then
washed and fixed, and the average number of sperm bound per egg was
determined. This graph represents data for seven individual
experiments, in which 20-30 eggs were examined per group per
experiment; the data are normalized with respect to the no peptide
control (defined as 100%; in these experiments, the average number of
sperm bound per control egg was 8.17 ± 0.54). The error
bars represent the S.E. The asterisks indicate
p < 0.05 as compared with the no peptide
control.
Disintegrin Domain on the
Binding of Recombinant Fertilin
to Eggs--
We conducted a series
of experiments to examine the effects of the fertilin
disintegrin
domain peptides on the binding of recombinant fertilin
to eggs.
There were two reasons for this. First, because multiple sperm ligands,
in addition to fertilin
, mediate sperm-egg membrane interactions
(9), there was the possibility that any of these peptides (a-1, a-2,
and/or a-3) could have an inhibitory effect on the binding of fertilin
, but this effect would not be robust enough to lead to a reduction in sperm binding. Therefore, we wanted to test whether any of the
peptides would inhibit the binding of our specific sperm ligand of
interest, fertilin
, to eggs. Second, we wanted to confirm that the
inhibitory effect of peptide a-3 on sperm-egg binding was attributable
to an inhibition of the binding of fertilin
and not of a different
sperm ligand.
to eggs (Fig. 5) mirrored
the results regarding sperm-egg binding (Fig. 4). Peptide a-3 reduced
the binding of recombinant fertilin
DCE to ~50% of control levels
(control being defined as the level of binding in the absence of
peptide) (Fig. 5). None of the other peptides affected recombinant
fertilin
DCE binding, with protein binding levels being comparable
with the levels of binding in the absence of any peptide. Peptide a-3
also reduced the binding of recombinant fertilin
D but did not have a significant effect on the binding of recombinant fertilin
CE (data
not shown). These observations suggest that synthetic peptide a-3
perturbs the interaction of the disintegrin-like domain of fertilin
with binding sites on the egg plasma membrane.
View larger version (14K):
[in a new window]
Fig. 5.
Effects of synthetic peptides a-1, a-2, and
a-3 on the binding of recombinant fertilin to
ZP-free eggs. ZP-free eggs were incubated in 0.5 mM of
the indicated synthetic peptide peptide (a-1, a-1s, a-2, a-2s, a-3, or
a-3s) for 60 min. The eggs were then incubated in 0.5 mg/ml of
recombinant fertilin
DCE for 60 min, after which the eggs were
washed and fixed. Egg-associated recombinant fertilin
protein was
detected using a luminometric immunoassay. The graph shows data
normalized with respect to the no peptide control (defined as 100%; in
these experiments, the average number of raw light units/10 s detected
per control egg was 33275.1 ± 6654.0). The graph represents the
results from three separate experiments, with 10-15 eggs examined per
experiment per group. The error bars represent the S.E. The
asterisks indicate p < 0.05 as compared
with the no peptide control.
disintegrin loop sequence (AQDECDVT) inhibited the binding of
recombinant fertilin
to eggs much more effectively (i.e.
lower IC50) than did synthetic peptides (4). Furthermore, in contrast to our experience with some batches of synthetic peptides, we have had no problems with toxicity of BAP-presented peptides on eggs
(4). We therefore used BAP-presented peptides to confirm our results
with synthetic peptides a-1 and a-3 (Fig. 4). We chose to examine the
a-1 sequence more closely because we were surprised that the a-1
synthetic peptide had no effect (Figs. 4 and 5). Disintegrin loop
peptide sequences inhibit disintegrin-mediated adhesion in studies of
other ADAMs (fertilin
(24-27), cyritestin (8, 28), ADAM9 (6, 29),
ADAM15 (15, 30), and ADAM23 (7)), and we therefore wanted to verify
that the a-1 amino acid sequence had no inhibitory effect on
recombinant fertilin
binding by using a BAP-presented peptide form
of the a-1 sequence. We surmised that the BAP-presented peptide form
could be more effective than the synthetic peptide form, based on our
studies of fertilin
(4). The experiments were designed as follows. BAP-presented peptide versions of a-1 and a-3 sequences (BAP-a-1 and
BAP-a-3, respectively) were tested at 40 µM because this
was the concentration with which we observed the maximal inhibitory effect with a BAP-presented peptide corresponding to the fertilin
disintegrin loop sequence (4). Two control BAP-presented peptides for
a-1 and a-3 were used: one with scrambled amino acid sequences
(BAP-a-1s or BAP-a-3s, respectively) and one with mutated sequences in
which two or three amino acids were changed (BAP-a-1 m or BAP-a-3m,
respectively). Mutated residues in BAP-a-1 m and BAP-a-3m were chosen
based on studies of the fertilin
disintegrin loop, in which the ECD
tripeptide, particularly the Asp residue, were determined to be
important for adhesive activity (4, 5). Therefore, the VCD in the a-1
sequence AEDVCDLP was changed to AEDVAALP in
BAP-a-1 m, and the ECD in the a-3 sequence DLEECDCG was
changed to DLEAAVCG. BAP, expressed from the parental
vector pHNAP without any modifications, was also used as a control.
binding to eggs (Table II) were similar
to the effects of the synthetic peptides (Fig. 5). Neither BAP-a-1 nor
its controls (BAP-a-1s and BAP-a-1 m) had a significant inhibitory
effect on recombinant fertilin
D binding (Table II), in agreement
with results with synthetic peptide a-1 on sperm-egg binding or
recombinant fertilin
binding (Figs. 4 and 5). (Please note that
recombinant fertilin
binding was not increased in either synthetic
or BAP-presented a-1 peptide, and we do not have evidence that the
sperm binding in the presence of synthetic peptide a-1 (Fig. 4) was due
to any stimulatory effect on sperm adhesion.) BAP-a-3 reduced the
binding of recombinant fertilin
D to eggs, and this effect appeared
to be concentration-dependent (Table II). At 20 µM, BAP-a-3 reduced the binding of recombinant fertilin
D to ~60% of control levels, and at 40 and 80 µM
BAP-a-3 reduced the binding of recombinant fertilin
D to ~50% of
control levels (Table II); all of these differences were statistically
significant as compared with control BAP. Some inhibitory effects were
observed with the scrambled control, BAP-a-3s, with reduced binding of
recombinant fertilin
D being observed with eggs treated with 40 and
80 µM BAP-a-3s (~60% of control; p < 0.05) (Table II). Such effects were not observed with the mutated
control, BAP-a-3m, implying that the effects of BAP-a-3s may be due to
the amino acid composition, with four of the eight amino acids being
acidic residues.
Effects of BAP-presented peptides on the binding of recombinant
fertilin D to eggs
binding. This assay utilized
fluorescent beads coated with the ligand of interest and then allowed
these ligand-coated beads to bind to ZP-free eggs (16). As shown in
Fig. 6, incubation of ZP-free eggs with 40 µM BAP-a-3 reduces the binding of recombinant fertilin
D-coated beads (Fig. 6F), as compared with untreated eggs
(Fig. 6B) or eggs incubated with control BAP (Fig.
6D). We estimated that the level of bead binding was reduced
by ~50%; this is consistent with the results using soluble
recombinant fertilin
D detected by the luminometric immunoassay
(Table II). BAP-a-3s appeared to have a comparable inhibitory effect
(Fig. 6H), whereas the levels of bead binding to
BAP-a-3m-treated eggs (Fig. 6J) appeared to be similar to
the levels of binding to the control eggs (Fig. 6, B and
D), consistent with the results presented in Table II from
the luminometric immunoassay for the binding of soluble recombinant fertilin
D.
View larger version (53K):
[in a new window]
Fig. 6.
Effects of BAP-presented peptides a-3,
a-3s, and a-3m on the binding of bead-immobilized recombinant
fertilin D. ZP-free eggs were incubated
in 40 µM of the indicated BAP-presented peptide (no
BAP-presented peptide, control BAP, BAP-a-3, BAP-a-3s, or BAP-a-3m) for
60 min. The eggs were then cultured in a 20-30-µl drop of Whitten's
medium containing a 0.02% suspension of fluorescent 0.2-µm beads
coated with recombinant fertilin
D and 40 µM of the
indicated BAP-presented peptide for 60 min. The eggs were then washed,
and bead binding to eggs was then viewed on a fluorescent microscope.
The egg treatments were as follows: no BAP-presented peptide
(A and B), control BAP (C and
D), BAP-a-3 (E and F), BAP-a-3s
(G and H), and BAP-a-3m (I and
J). Shown are the results of one experiment that was
repeated three times with similar results each time.
to Eggs--
We also examined whether the
a-3 sequence, as a synthetic peptide or a BAP-presented peptide, would
inhibit the binding of recombinant fertilin
. The amino acid
sequence of peptide a-3 (DLEECDCG) has homology to the sequence of the
disintegrin loop of fertilin
(AQDECDVT), including the ECD
tripeptide that has been shown to be important for mouse fertilin
function (4, 5). Therefore, we hypothesized that the a-3 sequence could inhibit the binding of recombinant fertilin
because of the presence of the ECD tripeptide.
with eggs (Table
III). The levels of binding in the
presence of 80 µM of BAP-a-3 and 80 µM of
BAP-
DL (positive control, with the sequence AQDECDVT, corresponding
to the disintegrin loop of fertilin
) were statistically similar.
The scrambled a-3s sequence, as a synthetic peptide (at 500 µM) and as BAP-a-3s (at 80 µM), inhibited recombinant fertilin
binding to eggs to ~52-58% of control
levels (Table III). BAP-a-3s had less of an effect when tested at 40 µM (reducing binding to ~77% of control levels). This
may indicate that the charge characteristics shared by peptides a-3 and
a-3s, both with four of the eight residues being acidic, affected the binding of recombinant fertilin
. In agreement with this, BAP-a-3m, with only two acidic residues, did not inhibit the binding of recombinant fertilin
to eggs (Table III).
Effects of peptides on the binding of recombinant fertilin to eggs
binding to eggs (Fig. 7).
BAP-a-3 significantly reduced the binding of recombinant fertilin
D-coated beads (Fig. 7F), as compared with untreated eggs
(Fig. 7B) or eggs treated with control BAP (Fig.
7D). BAP-a-3s appeared to have a modest inhibitory effect
(Fig. 7H), whereas the levels of bead binding to
BAP-a-3m-treated eggs (Fig. 7J) appeared to be similar to
the levels of binding to the control eggs (Fig. 7, B and
D), consistent with the results presented in Table III from
the luminometric immunoassay.
View larger version (49K):
[in a new window]
Fig. 7.
Effects of BAP-presented peptides a-3,
a-3s, and a-3m on the binding of bead-immobilized recombinant
fertilin D. ZP-free eggs were incubated
in 40 µM of the indicated BAP-presented peptide (no
BAP-presented peptide, control BAP, BAP-a-3, BAP-a-3s, or BAP-a-3m) for
60 min. The eggs were then cultured in a 20-30-µl drop of Whitten's
medium containing a 0.02% suspension of fluorescent 0.2-µm beads
coated with recombinant fertilin
D and 40 µM of the
indicated BAP-y for 60 min. The eggs were then washed, and bead binding
to eggs was then viewed on a fluorescent microscope. A,
C, E, G, and I show phase
contrast images; B, D, F,
H, and J show the partner fluorescent image. The
egg treatments were as follows: no BAP-presented peptide (A
and B), control BAP (C and D), BAP-a-3
(E and F), BAP-a-3s (G and
H), and BAP-a-3m (I and J). These are
the results of one experiment that was repeated three times with
similar results each time.
6 Integrin and Anti-CD9 Antibodies
on the Binding of Recombinant Fertilin
to Eggs--
Monoclonal
antibodies against the
6 integrin subunit (GoH3 (16))
and against the tetraspanin protein CD9 (KMC.8 (17) or JF9 (18)) have
been reported to inhibit sperm-egg binding during IVF, and there are
data that suggest these antibodies can perturb the binding of fertilin
to eggs. Because fertilin
is also a member of the ADAM family,
we hypothesized that GoH3 or KMC.8 could perturb the binding of
fertilin
. To test this hypothesis, we used the two different
binding assays described above to assess the binding of soluble
recombinant fertilin
D diluted in the culture medium using the
luminometric immunoassay and to assess the binding of recombinant
fertilin
D immobilized on fluorescent beads. The results from the
two assays were similar with GoH3. GoH3 (anti-
6) did not
have an effect on the binding of soluble recombinant fertilin
detected by the luminometric assay (Fig.
8A) and had a slight or
negligible inhibitory effect on the binding of recombinant fertilin
D immobilized on fluorescent beads as compared with the control
nonimmune IgG (Fig. 8B). Interestingly, however, different
results were obtained in the two different assays with KMC.8
(anti-CD9). KMC-8 did not inhibit the binding of soluble recombinant
fertilin
D (Fig. 8A) but had an inhibitory effect the
binding of bead-immobilized recombinant fertilin
D (Fig.
8B).
View larger version (33K):
[in a new window]
Fig. 8.
Effects of
anti- 6 (GoH3) and anti-CD9 (KMC.8)
monoclonal antibodies on the binding of recombinant fertilin
D. A, ZP-free eggs were incubated
in medium containing the indicated antibody (500 µg/ml of control rat
IgG or GoH3 or 50 µg/ml of KMC.8) or in medium in the absence of
antibody (No IgG) for 60 min. The eggs were then incubated
in medium containing 0.5 mg/ml of recombinant fertilin
D and the
indicated antibody for 60 min. The eggs were then washed and fixed.
Egg-associated recombinant fertilin
D was detected using a
luminometric immunoassay. The graph shows data normalized
with respect to the no antibody control (defined as 100%; in these
experiments, the average number of raw light units/10 s detected per
control egg was 7802.8 ± 692.7). The graph represents
the results from four separate experiments, with 10-15 eggs examined
per experiment per group. The error bars represent the S.E.
The differences were not statistically significant. B,
ZP-free eggs were incubated in the indicated antibody (500 µg/ml of
control rat IgG or GoH3; or 50 µg/ml of KMC.8) or in medium in the
absence of antibody (No IgG) for 60 min. The eggs were then
cultured in a 20-30-µl drop of Whitten's medium containing a 0.02%
suspension of fluorescent 0.2-µm beads coated with recombinant
fertilin
D and the indicated antibody for 60 min. The eggs were then
washed, and bead binding to eggs was then viewed on a fluorescent
microscope. The upper panels show phase contrast images; the
lower panels show the partner fluorescent image. The egg
treatments were as follows and as labeled: no IgG (first
column of images), control rat IgG (second column), 500 µg/ml GoH3 (third column), and 50 µg/ml KMC.8
(fourth column). These are the results of one experiment
that was repeated three times with similar results each time.
CE to eggs. KMC.8 did not affect the binding of
soluble recombinant fertilin
CE (data not shown). GoH3 decreased the
binding of soluble recombinant fertilin
CE very slightly, to ~75%
of levels observed to eggs treated with control IgG (data not shown).
However, we could not confirm this result using bead-immobilized
recombinant fertilin
CE. This may be because a modest inhibitory
effect (e.g. a 25% decrease, as was observed with the
luminometric immunoassay) in the level of bead binding is very
difficult to assess confidently.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
participates in sperm-egg adhesion during
fertilization and have identified a region of this domain that
partially mediates this adhesive activity. This region has the amino
acid sequence DLEECDCG and is in the N-terminal portion of the
disintegrin-like domain (amino acids 416-423, peptide a-3; Fig. 3). We
observe that a synthetic peptide with the a-3 sequence DLEECDCG
inhibits sperm-egg binding during IVF (Fig. 4) and that a synthetic
peptide and a BAP-presented peptide with the a-3 sequence inhibit the
binding of recombinant fertilin
to eggs (Figs. 5 and 6 and Table
II). Two other candidate peptides, including one corresponding to the
putative fertilin
disintegrin loop (peptide a-1), do not have these
effects. These data regarding fertilin
differ from those regarding
several other ADAMs. Adhesive events mediated by ADAMs such as fertilin
(24-27), cyritestin (8, 28), ADAM9 (6, 29), ADAM15 (31), and
ADAM23 (7) are inhibited by peptides corresponding to the sequences in
the putative disintegrin loops of these ADAMs. Additional studies of
the interactions of fertilin
with eggs (4) and of ADAM15 with
3-transfected CHO cells (30) suggest that the
disintegrin loops of these proteins are the key (and perhaps only)
adhesion-mediating sequences involved in these particular interactions.
Interactions of fertilin
with eggs is instead inhibited by a
peptide corresponding to the DLEECDCG region (Fig. 3; which we refer to
as the a-3 region, referring to its peptide name and the acronym
alternative adhesion-mediating motif in
fertilin
) of the fertilin
disintegrin-like domain.
contains an ECD sequence (Fig. 3), a
tripeptide that is also found in the putative disintegrin loops of
several ADAMs that function as cell adhesion molecules, such as
fertilin
(4, 5), ADAM9 (6), and ADAM23 (7). The ECD sequence in the
a-3 region of fertilin
is highly conserved in many ADAM family
members identified in eight mammalian species and in X. laevis, D. melanogaster, and C. elegans,
with 64 of 71 ADAMs having an ECD (42 of 71), or the similar sequences
DCD (4 of 71) or QCD (18 of 71) in the region aligning with the
fertilin
a-3 region. The putative disintegrin loop sequences are
similarly conserved, with 49 of 71 ADAMs having an ECD (38 of 71), a
DCD (7 of 71), or a QCD (4 of 71) tripeptide. Although it is not known at this time whether the a-3-like regions in other ADAMs play any role
in mediating adhesion or how these motifs are presented, the
conservation of this sequence in regions aligning with the fertilin
a-3 peptide is striking. Nevertheless, it is worth noting that the
critical sequence motif of the fertilin
disintegrin-like domain
remains to be definitively defined. Mutating the a-3 sequence DLEECDCG
to DLEAAVCG (a-3m) abolishes the ability of this sequence to
perturb interactions of recombinant fertilins
and
with the egg
plasma membrane, although the partial activity of CELDGDCE (a-3s)
implies that the amino acid composition or spacing (e.g. alternating acidic residues) could be a factor as well (Tables II and
III and Figs. 6 and 7). This differs from what is known about
disintegrin loop regions, with ECD motifs in fertilin
(4, 5) and
ADAM23 (7) and similar DCD, SCD, or ACD motifs in various forms of
ADAM15 and ADAM12 (15) being implicated in adhesive activity. The CD
residue pair does not appear to be sufficient to perturb the binding of
fertilin
or sperm, because the a-1 and a-2 sequences
(AEDVCDLP and DLPEYCDG, respectively) did not
have significant inhibitory activity (500 µM of a
synthetic peptide or 40 µM of a BAP-presented peptide;
Figs. 4 and 5 and Table II).
but also recombinant fertilin
to eggs. This agrees
with previous findings regarding the ECD tripeptide in fertilin
(4,
5) and augments these studies by demonstrating that an ECD tripeptide
in the context of a different amino acid sequence (DLEECDCG
instead of AQDECDVT) can perturb fertilin
binding to
eggs. In addition, the a-3 sequence is the first peptide shown to block
two different sperm ligands.
, the disintegrin-like domain and the cysteine-rich domain and/or
EGF-like repeat, participate in fertilin
-mediated adhesion of sperm
to the egg plasma membrane during fertilization (Fig. 2). This makes
fertilin
similar to ADAM12, which also potentially uses multiple
domains (the disintegrin-like domain (15) and the cysteine-rich domain
(14)) to mediate cell adhesion. To date, three different cysteine-rich
domains have been reported to participate in cell adhesion events:
those of fertilin
(Refs. 10 and 12 and this study), ADAM12 (14,
32), and atrolysin C, a snake venom metalloprotease that is closely
related to ADAM proteins (33). In contrast, the cysteine-rich domain of
fertilin
does not appear to have adhesive activity (10). Other ADAM cysteine-rich domains have not been examined to date.
-mediated adhesion and ADAM12-mediated adhesion. Moreover, the
utilization of two different regions in fertilin
and ADAM12 is
analogous to what is known about fibronectin. Fibronectin supports cell
adhesion via the
5
1 integrin through two
different domains, the canonical adhesion-mediating RGD tripeptide in
the tenth fibronectin type III repeat, and a synergy domain in the
ninth repeat, composed of sequence PHSRN and other amino acids (34,
35). Our data suggest that at least two regions of fertilin
are
involved in adhesion: the DLEECDCG a-3 region of the disintegrin-like
domain and some as yet unidentified portion(s) of the cysteine-rich
domain and/or EGF-like repeat. Furthermore, it is possible that the
disintegrin loop of fertilin
participates in fertilin
-mediated
adhesion, even though we did not observe any inhibition of sperm or
recombinant fertilin
binding to eggs with a-1 peptide (Figs. 4 and
5 and Table II), based on what is known about the fibronectin synergy domain. Although the ninth fibronectin type III repeat has no adhesive
activity on its own (36) and PHSRN peptides have no inhibitory activity
alone or in combination with RGD peptides (34), several residues in
this ninth type III repeat play a role in
5
1-mediated cell adhesion to fibronectin
(34, 35). BAP-a-3 did not completely inhibit the binding of recombinant fertilin
D even at concentrations up to 80 µM (Table I
and Fig. 6); this contrasts findings regarding fertilin
, in which
BAP-
DL (corresponding to the fertilin
disintegrin loop)
inhibited the binding of recombinant fertilin
D to near base-line
levels at concentrations of 30-40 µM (4). Thus, the a-3
region and the disintegrin loop and/or some other region(s) in the
fertilin
disintegrin-like domain may combine to have synergistic
activity in cell adhesion. In the future, it would be interesting to
investigate binding affinities of the disintegrin-like and
cysteine-rich domains and mutated versions thereof and to perform
further structure-function analyses to dissect how this sperm ligand
interacts with the egg membrane. Furthermore, because fertilin
can
form a heterodimer with fertilin
(23, 37, 38), the face of a
fertilin
-fertilin
dimer could present additional combinations
of binding sites, all of which could work synergistically to mediate
sperm-egg adhesion. Finally, the utilization of two different regions
in fertilin
raises the possibility that different receptors on the
egg surface may interact with different domains. This may be the case
with ADAM12, because the disintegrin-like domain appears to interact with the
9
1 integrin (15) and the
cysteine-rich domain appears to interact with a syndecan proteoglycan
(14, 32).
, we present data
indicating that an anti-CD9 antibody can perturb the binding of
recombinant fertilin
D immobilized on fluorescent beads (Fig. 8B). However, the anti-CD9 antibody had no effect on soluble
recombinant fertilin
D (i.e. diluted in culture medium
and detected by a quantitative luminometric immunoassay; Fig.
8A). The other antibody we tested, the anti-
6
integrin function-blocking antibody GoH3, did not dramatically affect
the binding of recombinant fertilin
D in either binding assay (Fig.
8). These data suggest that the GoH3 antibody does not significantly
perturb the ability of fertilin
to interact with the egg membrane
and that fertilin
immobilized on small beads interacts with a
KMC.8-sensitive binding site, whereas soluble fertilin
interacts
with a KMC.8-insensitive site. Although this dissimilarity of the
results with the KMC.8 antibody is perplexing, it may in fact be
providing some insights into how fertilin
interacts with the egg
plasma membrane, particularly in light of data on the molecular,
biochemical, and biophysical properties of complex adhesive events,
such as adhesion under flow conditions or adhesion during cell motility
(39, 40). The data from studies of these systems underscore the fact
that adhesion is an integrated process involving multiple binding
contacts (i.e. binding sites on individual molecules as well
as multiple types of molecules) and is affected by several biochemical
and biophysical parameters such as affinities, on and off rates, and ligand density and clustering. In this instance with fertilin
,
ligand density is very likely to be affected by immobilization of
ligand protein on beads, which could generate a microenviroment with a
relatively high concentration of ligand as compared with free ligand in
solution. The presentation of ligand on beads (0.2 µm in diameter)
also has the potential to mimic the presentation of fertilin
on the
mouse sperm head (~5 µm in length). Therefore, such considerations
are especially applicable to sperm-egg interactions (and thus to
fertilin
) because gamete adhesion events are likely to show
complexities that are somewhat similar to the complexities of adhesion
events of motile cells and cells under flow.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank William Hanna and Bayard Storey for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HD 37696, by a American Society for Reproductive Medicine and Organon, Inc. Research Grant in Reproductive Medicine, and by a Faculty Innovation Award from the Johns Hopkins University School of Public Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF167406 and U22056.
Supported by National Institutes of Health Training Grant HD07276.
§ Supported by National Institutes of Health Training Grant CA09110
¶ To whom correspondence should be addressed: Div. of Reproductive Biology, Dept. of Biochemistry and Molecular Biology, School of Public Health, Johns Hopkins University, 615 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-614-5557; Fax: 410-614-2356; E-mail: jpevans@jhsph.edu.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M101637200
2 X. Zhu and J. P. Evans, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ADAM, A disintegrin and A metallo- protease; EGF, epidermal growth factor; DCE, disintegrin/cysteine-rich/EGF-like repeat; CE, cysteine-rich/EGF-like repeat; ZP, zona pellucida; IVF, in vitro fertilization; MBP, maltose-binding protein; BAP, bacterial alkaline phosphatase; PCR, polymerase chain reaction.
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