Putative sperm fusion protein IZUMO and the role of N-glycosylation

IZUMO is the mouse sperm protein proven to be essential for fusion with eggs. It contains one immuno- globulin-like domain with a conserved glycosylation site within. In the present paper, we produced trans- genic mouse lines expressing unglycosylated IZUMO (N204Q-IZUMO) in Izumo1 / background. The expression of N204Q-IZUMO rescued the infertile phenotype of IZUMO disrupted mice, indicating glyco- sylation is not essential for fusion-facilitating activity of IZUMO. The N204Q-IZUMO was produced in tes- tis in comparable amounts to wild-type IZUMO, but the amount of N204Q-IZUMO on sperm was significantly decreased by the time sperm reached the cauda epididymis. These data suggest that glyco- sylation is not essential for the function of IZUMO, but has a role in protecting it from fragmentation in cauda epididymis

The mechanism of sperm-egg interaction and the involvement of IZUMO1 in fusion

Inoue, N., Ikawa, M., & Okabe, M. (2011). The mechanism of sperm-egg interaction and the involvement of IZUMO1 in fusion. Asian Journal of Andrology, 13(1), 81-87. doi:10.1038/aja.2010.7

Identification and Disruption of Sperm-Specific Angiotensin Converting Enzyme-3 (ACE3) in Mouse

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 2/2 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 AC

Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense

Background: Since Trypanosorna spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life -cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T brucei Hp-Hb complex receptor (TbHpHbR). Methods: Trypanosorna congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay. Results: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K,A) =2.1x10(-8) M) but free-Hp with low affinity (Kd = 2.2x10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome). Conclusion: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T congolense EMF-specific Hb receptor (TcEpHbR)

Liver-specific γ-glutamyl carboxylase-deficient mice display bleeding diathesis and short life span

Liver-Specific γ-Glutamyl Carboxylase-Deficient Mice Display Bleeding Diathesis and Short Life Span. Azuma K, Tsukui T, Ikeda K, Shiba S, Nakagawa K, et al. PLOS ONE. 2014. 9(2) doi:10.1371/journal.pone.008864