Both linear peptides (3,4,14,15) and existing
peptide mimics (19) of fertilinb and
cyritestin are modest inhibitors of sperm-egg binding with IC50’s
around 500 µM. We hypothesized that the modest inhibition
observed with linear peptides might be due to a monovalent binding
interaction that is of low affinity and, so we have been pursuing
the syntheses and analyses of two types of polyvalent inhibitor:
liposomes and polymers. Moreover, we felt that multivalent ligands
have the potential to act as sperm agonists and activate the egg
to develop. This type of activation could in turn block sperm
adhesion and fusion in trans, rather than acting through direct
competitive antagonism.
Liposomes presenting fertilinb peptides
are inhibitors of mouse in vitro fertilization (20). We undertook
a study of the relationship between IC50 and mole fraction of
fertilinb in the liposome, and the
size dependence of inhibition of fertilization to understand the
mechanism of their inhibition. Our results indicate that a small
number of multivalent contacts are required for efficient attachment
of inhibitor to receptor on the target membrane, and that, as
designed, the liposomes target the egg membrane but not the sperm
membrane (21). Unlike monomeric inhibitors which saturate inhibition
at approximately 70%, the liposomes completely inhibit sperm egg
binding. This observation and the correlation of concentration
dependence of inhibition with liposome size demonstrate that the
liposomes physically block access of the sperm to the egg membrane
thereby preventing sperm binding to all egg receptors not just
the fertilinb receptor.
We undertook the synthesis of multivalent peptide mimics that
allow control over not only the surface concentration and type
of ligands presented, but also their density, to further investigate
the role of polyvalency in the sperm-egg adhesion process. We
used ring-opening metathesis polymerization to prepare oligopeptide
polymers (Scheme 1) (22). These polymers are kinetically stable
in the egg culture conditions, and we can synthesize polymers
with as many as 100 pendent oligopeptides. Thus, in addition to
testing the potency of the polymers, we were able to explore their
mechanism of inhibition. At present, the majority of our work
has focused on the first generation polymer 1-10.
The fertilinb peptide norbornyl polymer 1-10 is a significantly
better inhibitor of sperm-egg adhesion than its monomeric peptide
counterpart. The scrambled sequence polymer 2-10 showed no inhibition
at 400 µM. This suggests that multivalent interactions of
the polymer are important for inhibition and that they are not
behaving as non-specific anionic detergents. However, just like
the monomer, inhibition saturates at approximately 70%. This fits
with our postulate that there is more than one receptor-ligand
interaction required for sperm-egg binding and that the peptide
sequences of these monomers and polymers only block one. The liposomes
are 40 nm in diameter as compared to the 6 nm theoretical span
of the extended 10-mer polymer backbone. If IC50’s are compared
by calculating the number of molecular complexes (i.e., liposome
or polymer) per mL at half-maximal inhibition, we observe that
30-fold more polymers are required than liposomes. This comparison
demonstrates that a small number of multivalent contacts are required
for efficient attachment of inhibitor to receptor on the target
membrane, but the physical size of the inhibitory complex contributes
to steric blocking of sperm binding to the egg plasma membrane.
The difference between liposome and polymer behavior suggests
that binding the liposome to the surface of the egg blocks sperm
access to an unidentified receptor or receptors, whereas, the
short oligopeptide polymers only competitively block fertilinb
receptor. These experiments highlight the value of comparing two
different types of polyvalent inhibitor to elucidate the importance
of individual receptor–ligand interactions. We are currently examining whether polymer 1-10 is capable of parthenogenically
activating the egg.
Concentration dependence of inhibition by number
of eggs fertilized (FR). Solid squares, fertilinb
polymer 1-10; open squares, fertilinb monomer ; open circles, 1 mol%
fertilinb liposome. Fertilization rate is relative to a buffer
control in which 76% of the eggs were fertilized and an average
of 1.6 sperm were fused per egg. Error bars are s.e.m. where n
= 2-6, 30-75 eggs.
To address the receptor specificity, we developed a synthesis
of a fluorescently labeled polymer in order to directly visualize
whether the fertilinb polymer 1-10
binds to the egg surface, and to assess which receptors it might
be ligating. Our approach was to make a block copolymer of fertilinb
peptides and N-hydroxysuccinimide esters. This synthetic approach
allows us to incorporate more or less fluorophores as needed to
detect the polymer, simply by varying the number of equivalents
added in the synthesis. The fluorophore is at the terminus of
the polymer by virtue of adding the N-hydroxysuccinimide norbornene
monomer first. The fluorophore is added after polymerization,
and any nucleophilic fluorophore, or other type of tag, may be
added, depending on the use at hand. We see that the polymer cleanly
labels the egg plasma membrane and receptor colocalization experiments
are in progress.
