Structural studies of proteins essential for fertilization

Where do babies come from? Even though it sounds like a fairly obvious question, it took
more than 200 years to discover that sperm, whose first observation dates back to the 17th
century, can penetrate into the oocyte and fuse with it. Interestingly, this initial study was
carried out in starfish, and since then, scientists have addressed the fascinating question
of how gamete recognition functions using very different model systems.

Egg-sperm binding is a species-specific event, and it is thought to require binding proteins
that are exposed on the surface on the egg and sperm. In this thesis, we focus our
attention on how egg coat subunits are involved in this process and investigate it using
X-ray crystallography and biochemistry.



The mammalian egg coat, also known as Zona Pellucida (ZP), is a thick extracellular
matrix that surrounds the egg and is made of glycoproteins (known as ZP1-4) that
assemble into long cross-linked filaments. Although ZP3 has been identified as a sperm
receptor, it remains unclear as to how it interacts with sperm at molecular level. Paper I
explores the potential role of sperm protein 56 (sp56) as a receptor for the egg coat protein
ZP3. Its putative role as a ZP3-binding partner has been previously assessed by several in
vitro assays using soluble proteins. Here, we express the recombinant form of hamster
sp56 and attempt to reconstitute a hetero-complex with recombinant ZP3. Sp56 is secreted
as a homo-hexamer held together by intramolecular disulfides. Considering the high avidity
conferred by the homo-oligomeric state of the glycoprotein, the inability to observe any
interaction to ZP3 by either pull-down or Far western blotting (Far WB) indicates that
very-low affinity binding events might mediate egg-sperm interaction.

Different from the mammalian scenario, egg and sperm proteins in marine invertebrates
are known to bind more tightly. Paper II sheds light on the event occurring when gametes
first meet using abalone as a model system. We discover that egg coat functional units in
mammals and invertebrates are structurally conserved and, remarkably, despite millions of
years of evolution they rely on a common ZP-N fold domain to accomplish their functions.
Crystallographic studies on individual subunits of the egg coat receptor VERL (vitelline
envelope receptor for lysin) in complex with the sperm protein lysin, provides the first
snapshot of egg-coat recognition and clarifies how species-specificity is maintained. By
combining structural and biochemical data, the paper proposes the mechanistic model of
how sperm penetrates the egg coat at the molecular level.



For structural studies of egg coat glycoproteins, it is essential to produce correctly folded
material that is stable, highly homogeneous and is secreted in large amounts. To achieve
this, we fuse glycoproteins to maltose binding protein (MBP) and describe in Paper III the
advantages of such a method using mammalian and insect cell expression systems. The
study herein shows how fusion to MBP boosts the expression of six challenging
glycoproteins that are poorly secreted if unfused. MBP not only improves crystallization but
provide initial phasing information for structural determination by molecular replacement.

Given the relevant role of egg coat subunits in fertilization, we investigate in Paper IV their
functional relationship to two specular aspects of reproductive health. The study provides
detailed information on how an anti-ZP-reagent recognizes its epitopes, and investigates
how mutations in ZP genes relate to female infertility. By doing so, our findings further
strengthen the importance of ZP in the development of novel contraceptive strategies and
provide a better understanding of the impact of ZP mutations on human fertility