Background: IZUMO1 is the only sperm protein which is proven to be essential for sperm-egg fusion. However, the IZUMO1 is a structurally simple protein with single Ig domain and seems not to include either a "fusogenic peptide" or a fusion machinery domain. This led us to assume the existence of an IZUMO1-interacting protein(s) which makes a functional fusion machine interacting with IZUMO1.
Methodology/principal findings: We produced a transgenic mouse line which expresses His-tagged IZUMO1 in the Izumo1(-/-) genetic background. After solubilization of sperm membranes, we purified His-tagged IZUMO1 using anti-His affinity chromatography and found a protein that interacts with IZUMO1. After being separated on SDS-PAGE gel, the IZUMO1-interacting protein was subjected to LC-MS/MS analysis and from the partial fragments, we identified the protein as ACE3. We raised the antibody against ACE3 and found that ACE3 is localized on the acrosomal cap area as in the case of IZUMO1. However, ACE3 disappeared from sperm after acrosome reaction while IZUMO1 remained on sperm. In order to investigate the role of ACE3 in vivo, we generated Ace3-deficient mice by homologous recombination and examined the fertilizing ability of the males. Unexpectedly, the male mice showed no defect in fertilizing ability in in vivo or in an in vitro fertilization system.
Conclusions/significance: We identified an IZUMO1-interacting protein in sperm, which we identified as testis specific ACE homologue ACE3. We produced an Ace3 disrupted mouse line, and found the localization of IZUMO1 spread in a little wider area on sperm, but the elimination of ACE3 did not result in a loss of sperm fertilizing ability, differing from the case of ACE disruption.