title: Residues SFQ (173-175) in the large extracellular loop of CD9 are required for gamete fusion.
abstract: Gamete fusion is the fundamental first step initiating development of a new organism. Female mice with a gene knockout for the tetraspanin CD9 (CD9 KO mice) produce mature eggs that cannot fuse with sperm. However, nothing is known about how egg surface CD9 functions in the membrane fusion process. We found that constructs including CD9's large extracellular loop significantly inhibited gamete fusion when incubated with eggs but not when incubated with sperm, suggesting that CD9 acts by interaction with other proteins in the egg membrane. We also found that injecting developing CD9 KO oocytes with CD9 mRNA restored fusion competence to the resulting CD9 KO eggs. Injecting mRNA for either mouse CD9 or human CD9, whose large extracellular loops differ in 18 residues, rescued fusion ability of the injected CD9 KO eggs. However, when the injected mouse CD9 mRNA contained a point mutation (F174 to A) the gamete fusion level was reduced fourfold, and a change of three residues (173-175, SFQ to AAA) abolished CD9's activity in gamete fusion. These results suggest that SFQ in the CD9 large extracellular loop may be an active site which associates with and regulates the egg fusion machinery.
authors.affiliation: Department of Cell Biology and Human Anatomy, School of Medicine, University of California Davis, Davis, CA 95616, USA. gzhu@ucdavis.edu
authors.forename: Guo-Zhang
authors.initials: GZ
authors.lastname: Zhu
authors.forename: Brent J
authors.initials: BJ
authors.lastname: Miller
authors.forename: Claude
authors.initials: C
authors.lastname: Boucheix
authors.forename: Eric
authors.initials: E
authors.lastname: Rubinstein
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Liu
authors.forename: Richard O
authors.initials: RO
authors.lastname: Hynes
authors.forename: Diana G
authors.initials: DG
authors.lastname: Myles
authors.forename: Paul
authors.initials: P
authors.lastname: Primakoff
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D002459:Cell Fusion; D005110:Extracellular Space; D005260:Female; D005854:Germ Cells; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008845:Microinjections; D009865:Oocytes / Q000502:physiology; D010063:Ovum / Q000502:physiology; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology; D060245:Tetraspanin 29
chemical_list: D015703:Antigens, CD; C554869:CD9 protein, human; C554870:Cd9 protein, mouse; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D060245:Tetraspanin 29; D005982:Glutathione Transferase