Functional role of feline zona pellucida protein 4 trefoil domain: a sperm receptor or structural component of the domestic cat zona pellucida?
The trefoil domain (TFD) is a protein structure characterized by six cysteines, which form a typical three-loop structure by three disulphide bridges. It is assumed that two of these loops generate a hydrophobic groove, which could be a binding site for carbohydrate residues or proteins. The zona pellucida (ZP) protein, ZP4, contains such a TFD. The carbohydrate-/protein-binding property of TFD allows us to assume a potential sperm receptor function of this domain. Additionally, gastrointestinal trefoil peptides are stable against proteases; therefore, a structural role of TFD within the ZP might also be possible. We were able to show that the synthesized and natural folded feline TFD (fTFD) expresses the typical protease resistance that vanished under reducing conditions and after substitution of cysteine residues within the peptide. Furthermore, an antibody directed against the first loop of fTFD was almost unable to bind to intact in vitro mature cat oocytes. Pre-incubation of oocytes in the reducing agent (DDT), however, improved antibody binding substantially. Therefore, we suggest structural masking of the fTFD domain within the intact ZP. An interaction between fTFD and feline sperm cells was examined using several methods, including immunocytochemistry, immunoelectron microscopy, co-immunoprecipitation and far western blot, but we found no indication for an involvement of TFD in the primary sperm binding to the ZP. To summarize, there is increasing evidence that the TFD of fZP4 has a structural rather than a sperm-binding function.
Braun
B C
BC
Department for Reproductive Biology, Leibniz-Institute for Zoo and Wildlife Research, Berlin, Germany. braun@izw-berlin.de
Ringleb
J
J
Waurich
R
R
Viertel
D
D
Jewgenow
K
K
D000818:Animals; D002415:Cats / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D013094:Spermatozoa / Q000502:physiology*; D015044:Zona Pellucida / Q000378:metabolism*
D008562:Membrane Glycoproteins