Structural and functional insights into IZUMO1 recognition by JUNO in mammalian fertilization

Abstract

Sperm–egg fusion is the critical step in mammalian fertilization, and requires the interaction between IZUMO1 on the sperm surface and JUNO (also known as folate receptor (FR) 4 or IZUMO1R) on the egg surface. Whereas other FRs bind and uptake folates, JUNO binds IZUMO1 and establishes the cell–cell adhesion. However, the mechanism of IZUMO1 recognition by JUNO has remained elusive. Here we report the crystal structure of mouse JUNO, at 2.3 Å resolution. A structural comparison of JUNO with the FRs revealed that JUNO and the FRs have similar overall structures, but JUNO lacks the folate-binding pocket, thereby explaining the inability of JUNO to bind folate. Further complementation of Juno knockout eggs with mutant Juno messenger RNAs revealed that the conserved, surface-exposed tryptophan residue of JUNO is required for sperm binding and fertilization. Our structure-based in vivo functional analyses provide a framework towards a mechanistic understanding of mammalian gamete recognition.

Structural insights into tetraspanin CD9 function

“…Gene complementation experiments using Cd9 deficient eggs were performed basically according to the previous study 68 . Immature, germinal vesicle eggs were collected from the ovaries of wild-type B6D2F1 or Cd9 KO female mice (10-20 weeks old), 46 h after the injection of anti-inhibin antibody solution (CARD HyperOva, Kyudo).…”

“…After a quick wash in kSOM medium, the eggs were subjected to confocal imaging. Confocal images were acquired as described 68 . Experiments were repeated at least three times for each batch.…”

The hallmarks of cell-cell fusion

“…In mammalian spermegg fertilization, the adhesive complex formed between IZUMO1 in the sperm and JUNO (IZUMO1R) in the egg is essential for fertilization (Bianchi et al, 2014;Inoue et al, 2005), while sequence variations at the IZUMO-JUNO complex interface have been proposed to control recognition specificity between different mammalian species (Aydin et al, 2016). The structural biology of such adhesion and recognition machineries reveals how fusion-fated cells maintain close separation (Aydin et al, 2016;Kato et al, 2016;Nishimura et al, 2016;Ohto et al, 2016;Raj et al, 2017), a prerequisite before initiation of membrane fusion. However, there is weak support for the idea that adhesive complexes themselves are mechanically involved in membrane fusion.…”

When sperm meets egg

“…The structures of Juno from mice 7,8 and the current studies reveal that the protein has an almost identical fold to that of folate receptors 9,10 : globular, stabilized by eight disulfide bonds (S-S) and with a deep, ligand-binding pocket. But several key amino-acid residues in Juno's ligand-binding pocket differ from those of the folate receptors, consistent with the fact that Juno cannot bind folates 4 .…”