Molecular basis of egg coat cross-linking sheds light on ZP1-associated female infertility

Abstract

Mammalian fertilisation begins when sperm interacts with the egg zona pellucida (ZP), whose ZP1 subunit is important for fertility by covalently cross-linking ZP filaments into a three-dimensional matrix. Like ZP4, a structurally-related component absent in the mouse, ZP1 is predicted to contain an N-terminal ZP-N domain of unknown function. Here we report a characterisation of ZP1 proteins carrying mutations from infertile patients, which suggests that, in human, filament cross-linking by ZP1 is crucial to form a stable ZP. We map the function of ZP1 to its ZP-N1 domain and determine crystal structures of ZP-N1 homodimers from a chicken homolog of ZP1. These reveal that ZP filament cross-linking is highly plastic and can be modulated by ZP1 fucosylation and, potentially, zinc sparks. Moreover, we show that ZP4 ZP-N1 forms non-covalent homodimers in chicken but not in human. Together, these data identify human ZP1 cross-links as a promising target for non-hormonal contraception.

Mammalian egg coat modifications and the block to polyspermy

“…In relation to these considerations, it will be essential to gain much more detailed information on the structure of the egg coat than what is currently known. Obtaining this knowledge will not only depend on further X-ray crystallographic studies of isolated ZP subunits or domains thereof, an approach that already yielded precious information on ZP1 (Nishimura et al, 2019), ZP2 (Bokhove et al, 2016), and ZP3 (Han et al, 2010;Monné et al, 2008), as well as a first example of how sperm recognizes the egg coat in an invertebrate model of FAHRENKAMP ET AL.…”

“…In contrast with these findings on mouse fertilization, residues such as mannose, fucose, GlcNAc, and the sialyl-Lewis X sequence [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc] of N-and O-glycans have been suggested to play a role in human egg-sperm binding (Miranda et al, 1997;Oehninger, Patankar, Seppala, & Clark, 2009;Pang et al, 2011). The importance of these chemical moieties may explain the different sperm-binding properties of affinity-purified native human ZP proteins (Chiu et al, 2008) and a recent crystallographic analysis of chicken ZP1-N1 homodimers (Nishimura et al, 2019) suggested that core fucosylation of a conserved N-glycan that immediately precedes the intermolecular disulfide of the protein may favor a specific conformation of the ZP filament cross-links.…”

“…This raises the possibility that post-fertilization defucosylation of ZP1 contributes to hardening of the egg coat (Nishimura et al, 2019).…”

“…Biochemical and electron microscopy (EM) analysis of ZP filament material treated with reducing agents or chymotrypsin indicated that, by forming intermolecular disulfide bond(s), ZP1 acts as a cross-linker of individual ZP filaments (Greve & Wassarman, 1985). This suggestion was supported by a recent crystallographic study of the ZP1 cross-link that, together with mutagenesis experiments, revealed that a single ZP-N1 domain Cys conserved among species is essential for ZP1 homodimerization in chicken, mouse, and human (Nishimura et al, 2019).…”

“…Whereas ZP subunit secretion depends on the EHP (Jovine et al, 2004;Zhao et al, 2003), F I G U R E 1 Scheme of the domain architecture of human and mouse ZP components. The positions of the conserved ZP1-specific Cys essential for filament cross-linking (Nishimura et al, 2019) and the post-fertilization cleavage site of ZP2 important for ZP hardening (Gahlay, Gauthier, Baibakov, Epifano, & Dean, 2010) are marked by magenta arrowheads and black arrows, respectively. Dark gray rectangle, SP; light yellow diamond, trefoil/P domain; red rectangle, CFCS; dark yellow rectangle, EHP; brown rectangle, transmembrane domain.…”